Much interest has focused on how stem cells are maintained by intrinsic self-renewal capacity and environmental signals in specialized niches. However, less is known regarding how niches are maintained. We show here that ependymal cells and astrocytes have considerable phenotypic plasticity and can replace each other. The finding that the same signaling pathways, EphB and Notch, reciprocally regulate ependymal and astrocytic phenotypes is intriguing. It may provide an ingenious way to ensure the maintenance of the stem cell niche, as the modulation of these signaling pathways in the situation of a lesion provides a safeguard for controlling the cell composition of the niche. Extensive remodeling occurs in the ventricle wall stem cell niche in the adult brain in response to injury (Barnabé-Heider et al., 2008
; Carlén et al., 2009
; Conover et al., 2000
; Kuo et al., 2006
; Luo et al., 2008
). The neuronal production is rapidly regained after insults, demonstrating the capacity of the stem cell lineage and niche to regenerate (Doetsch et al., 1999b
). It was suggested, based on the lack of proliferation of forebrain ependymal cells, that the increase in the number of astrocytes in the ependymal layer after a lesion was the result of subventricular zone astrocytes migrating towards the ventricle lumen (Luo et al., 2008
). However, ependymal cells are, in spite of their quiescence, rather plastic and respond to stroke by giving rise to astrocytes and neuroblasts (Carlén et al., 2009
). Ependymal cells in the adult spinal cord are even more plastic and display neural stem cell properties (Barnabé-Heider et al., 2010
; Meletis et al., 2008
). We foundnd that most astrocytes intercalated within the ependymal layer derive from ependymal cells. However, astrocytes do also enter the ependymal layer, but the majority of these cells loses astrocytic features and differentiates to ependymal cells with expression of typical molecular markers, a cuboidal shape and multiple cilia. Both the conversion of astrocyte to ependymal cell and vice versa occurs without cell division. Thus, the main subventricular zone niche cells show a high degree of plasticity and are phenotypically mutually interconvertible.
Ependymal cells and subventricular zone astrocytes both originate from radial glial cells (Spassky et al., 2005
), which act as neural stem cells in the developing central nervous system. Many genes associated with neural stem/progenitor cells, including nestin, Sox2, Prominin-1/CD133 and Notch-1, are commonly expressed in ependymal cells and subventricular zone astrocytes (Doetsch et al., 1997
; Ellis et al., 2004
; Ferri et al., 2004
; Johansson et al., 1999
; Sakakibara et al., 2002
; Weigmann et al., 1997
). The maintenance of genetic programs similar to that of a common precursor may contribute to the fate plasticity of ependymal cells and astrocytes.
The ependymal layer was thought to be incapable of regeneration and that loss of this niche cell type would be permanent. This is indeed the case after loss of larger patches of ependymal cells (Carlén et al., 2009
; Kuo et al., 2006
). A previous report suggested that subventricular zone astrocytes could replace lost ependymal cells after minor lesions (Luo et al., 2008
), which we here confirmed by genetic fate mapping. We never saw groups of astrocyte-derived ependymal cells, but they were always present as individual cells within the ependymal layer. It is unclear why ependymal cells can be replaced after small but not large lesions, but may suggest that neighboring ependymal cells are required for successful generation of new ependymal cells. It is noteworthy in this context that the ephrin/Eph contact is restricted to direct cell contacts, since both the receptor and ligand are membrane bound, and that ependymal cells express both ephrin-B ligands and EphB receptors (Figure S3
). Our finding that EphB2 signaling is required for maintaining the ependymal cell phenotype suggests that contact between ependymal cells may promote EphB2 signaling and acquiring the full ependymal phenotype. Moreover, ependymal cells express both Notch1 and its cognate ligands Delta-like 1 and Jagged-1 and constitutive Notch signaling is also required for ependymal cell maintenance (Carlén et al., 2009
). Thus, contact with neighboring ependymal cells may be required for attaining and/or maintaining the ependymal cell phenotype, which could explain why small, but not large, lesions are repaired.
We find that Notch signaling positively regulates EphB2 expression in ependymal cells. Regulating the expression of EphB2 is an important part of the effect of Notch signaling, as ectopic expression of EphB2 can rescue the loss of ependymal phenotype seen after ablation of Notch signaling. However, it is clear that EphB2 is not the sole effecter of Notch signaling in this context, as ablation of Rbpj results in ependymal cell progeny entering the cell cycle and subsequently differentiating to neurons (Carlén et al., 2009
), neither of which is seen after inhibition of EphB2 signaling. Notch signaling is a well-documented determinant of neuron-glial fate choice. After stroke, Notch signaling is reduced in many ependymal cells, and ependymal cells give rise to both astrocytes and neuroblasts. When Notch signaling is completely blocked by deletion of Rbpj in ependymal cells, they do not give rise to any astrocytes but only neurons, demonstrating the pivotal role for Notch signaling in the fate of ependymal cell derived cells (Carlén et al, 2009
). We find that Notch signaling intensity is unaltered in the neuraminidase lesion model (Figures S6A-6C
), providing a likely explanation for why the ependymal progeny fail to give rise to neurons in this paradigm and is restricted to the astrocytic lineage.
The role of ephrins and Eph receptors has been most extensively studied in cell migration and axon guidance (Egea and Klein, 2007
; Pasquale, 2008
). As a migrating cell will be exposed to new molecular environments, it is often challenging to distinguish whether an effect of modulated Eph signaling may be direct or secondary to altered cell positioning. That Eph signaling has other direct effect than regulating cell positioning is clear from studies of intestinal progenitor cells, where the signaling pathways regulating cell migration and proliferation diverge at the level of the receptor protein (Genander et al., 2009
). We find in the current study that the ephrin-Eph interaction in the lateral ventricle wall regulate both cell phenotype as well as positioning with regard to the ependymal layer or subventricular zone. It is difficult to completely dissociate these effects, but the finding that ependymal cells differentiate to astrocytes within the ependymal layer appears difficult to explain as secondary to a cell migration effect of EphB signaling. Regulation of stem cell niche plasticity is a novel role for this family of tyrosine kinase receptors. Ephrins and Eph receptors are expressed in many adult stem cell systems (Holmberg et al., 2006
), suggesting that they may regulate niche function and plasticity in several organs.