(A) Experimental strategy for lineage tracing of astrocytes. Transgenic mice expressing tamoxifen inducible Cre recombinase under the control of the connexin30 promoter (Cx30-CreER) were crossed with R26R-YFP reporter mice. (B) Distribution of recombined cells in the subventricular zone and brain parenchyma after 5 days injection of tamoxifen. (C-F) Recombined cells are positive for the astrocytic markers GFAP (C) and glutamine synthetase (GluS, D) and some for the proliferation marker Ki67 (E), but none the ependymal cell marker S100ß (F). (G-L) Astrocytes give rise to ependymal cells after a neuraminidase lesion. A recombined cell expressing S100ß but not GFAP is integrated in the ependymal layer (arrowheads in G-J). (K and L) Whole mount analysis reveals the localization of gamma-tubulin on the apical surface of a recombined astrocyte-derived cell (arrowhead), as in neighboring non-recombined ependymal cells. Scale bars indicate 200 μm in (B), 10 μm in (F), (J) and (K).

(A) Experimental outline for assessing the effect on astrocytes of blocking the EphB/ephrin-B interaction. (B-E) In Fc-infused mice, YFP-positive recombined astrocytes do not display the ependymal cell marker S100ß or rootletin (B and C), while in EphB2-infused mice recombined cells express S100ß together with rootletin (D and E). (C and E) En face views of the ependymal layer in Fc (C) and EphB2-Fc-infused mice (E). Basal bodies of cilia in ependymal cells are visualized with an anti-rootletin antibody. (F-J) Fate conversion of ependymal cells into astrocytes by blocking of EphB/ephrin-B interactions. (F) Ependymal cells were labeled with adeno-Cre or adeno-GFP virus, and Fc or EphB2-Fc soluble protein was infused into the lateral ventricle one week later. (G and H) In an animal infused with Fc protein, S100ß expression is retained in GFP-positive ependymal cells (G), and GFAP expression is not detected (H). (I and J) In an animal infused with EphB2-Fc protein, some GFP-labeled ependymal cells have lost S100ß expression and have delaminated from the ependymal layer (arrowheads in I), and express GFAP (arrowheads in J). The luminal side of the ependymal layer is indicated by a broken line in (H). The scale bars indicate 10 μm in (B) and (G).

(A-G) Immunoreactivity of EphB2 in ependymal cells after neuraminidase treatment in R26R-YFP mice. Ependymal cells were labeled by injection of an adeno-Cre virus into the lateral ventricle. Compared to control (A-C), EphB2 expression is reduced in recombined ependymal cells 2 weeks after neuraminidase treatment (D-F). Note that GFAP expression is increased in the recombined cells (arrowhead). (G) Quantification of EphB2-immunoreactivity reveals a decrease after a neuraminidase lesion. (H-K) Overexpression of EphB2 inhibits astrocytic conversion of ependymal cells. (H) Experimental procedures of EphB2 overexpression combined with neuraminidase treatment. Electroporation of EphB2-expression vector partly blocks the transition of ependymal cells to astrocytes (I-K). Bars show mean + SD, each for n=3 mice. *=p<0.05, Student's t-test. Scale bar represents 10 μm in (A) and 20 μm in (J),

(A-F) The ventricle wall appears largely intact after recombination in Cx30-CreER;Rbpj^loxP/+ mice. (G-L) Recombination in Cx30-CreER;Rbpj^loxP/loxP mice results in the apperance of astrocyte derived ependymal cells (arrows) expressing S100ß, EphB2 and CD133. The images in (A-D, G-J) are from two months and the images in (E, F, K and L) are from 3 months after induction of recombination by injection of tamoxifen. The number of recombined ependymal cells per section was 0.12 ± 0.17 in Cx30-Cre;Rbpj^loxp/+ mice (total 572 recombined cells analyzed), 4.3 ± 2.5 in Cx30-Cre;Rbpj^loxp/loxP mice (total 750 recombined cells analyzed). Scale bar represents 20 μm in (L).

(A) Illustration of the strategy for genetic fate mapping of ependymal cells. (B-H) Recombined cells are distributed along the surface of the lateral wall (B), and labeled with S100ß (C) and CD133 (D), but not with GFAP (E), glutamine synthetase (GluS, F), DCX (G) or Ki67 (H). (I and J) Expression of the astrocyte markers GFAP (I) and GluS (J) in ependymal cells after neuraminidase treatment. Arrowheads in (I) indicate a recombined cell expressing both S100ß and GFAP and arrowheads in (J) indicate GluS expressing ependymal cell-derived astrocytes. (K and L) Delamination of ependymal cell-derived cells from the ependymal layer after a neuraminidase induced lesion. Arrowheads point to GFAP-positive recombined cells in the subventricular zone (K) and a CD133-negative cell (L). The luminal side of the ependymal layer is indicated by a broken line in (H). Scale bars represent 200 μm in B, 10 μm in (C), (I) and (K).

(A) Illustration of the experimental procedure. After labeling ependymal cells with adeno-GFP virus, plasmid vectors were electroporated into the lateral wall. (B and C) GFP expression by adenovirus transduction (B) and ß-galactosidase (ß-gal) expression by plasmid electroporation (C). (D-G) In a control animal electroporated with ß-gal expression vector, a cell which is double-positive for GFP and ß-gal does not express GFAP (open arrowheads). (H-K) In an animal electroporated with a vector expressing EphB2^Y604F,Y610F together with the ß-gal expression vector, a cell which is double-positive for GFP and ß-gal expresses GFAP in the ependymal layer (white arrowheads). (L-O) In an animal electroporated with a vector expressing ephrin-B2ΔC together with the ß-gal expression vector, a cell which is double-positive for GFP and ß-gal does not express GFAP (open arrowheads). (P-S) Analysis of the ependymal layer in EphB2 or ephrin-B2-deficient mice. There are no GFAP and S100ß co-expressing cells in the ependymal layer of wild type mice (WT, P), but they are present (indicated by arrows) in both EphB2^-/- (Q) and EphB2^lacZ/lacZ mice (R). The ventricle wall appears indistinguishable from the WT in ephrin-B2^lacZ/6YFdV mice (S). The luminal side of the ependymal layer is indicated by broken lines. Scale bars indicate 200 μm in (B) and 10 μm in (D).

(A and B) In a mouse heterozygous for a conditional allele of Rbpj (Rbpj^loxP/+), EphB2 expression is maintained in ependymal cells after an adeno-Cre virus injection (indicated by white arrowheads). (C and D) In a mouse homozygous for the conditional allele of Rbpj (Rbpj^loxP/loxP), a decrease in EphB2 expression is evident 1 week after injection of an adeno-Cre virus (indicated by a white bracket). (E) Quantification of EphB2-immunoreactivity in ependymal cells. (F and G) Expression of EphB2 in a control animal electroporated with a GFP expression vector. (H and I) High expression of EphB2 was detected in electroporated cells 3 days after electroporation of NICD. (J) Quantification of EphB2-immunoreactivity in ependymal cells.
(K, L and Q) In mice electroporated with a GFP expression vector, the majority of GFP-expressing cells are S100ß-immunoreactive ependymal cells (white arrowheads). (M, N and Q) In mice electroporated with a dominant negative Rbpj expression vector (dnRbpj-ires-GFP), the number of S100ß-positive GFP-labeled cells is decreased to 55.9%. Open arrowheads indicate GFP-positive, S100ß-negative cells. (O, P and Q) In mice electroporated with dnRbpj-ires-GFP and EphB2 expression vectors, the number of S100ß-positive GFP-labeled cells is restored to 89.3%. White arrowheads indicate GFP and S100ß double-positive cells. The bars in (E, J, and Q) show mean + SD. *=p<0.05, **=p<0.01, Student's t-test. Scale bars represent 10 μm in (A) and 20 μm in (K).