Forebrain ependymal cells are Notch-dependent and generate neuroblasts and astrocytes after stroke

Marie Carlén, Konstantinos Meletis, Christian Göritz, Vladimer Darsalia, Emma Evergren, Kenji Tanigaki, Mario Amendola, Fanie Barnabé-Heider, Maggie S.Y. Yeung, Luigi Naldini, Tasuku Honjo, Zaal Kokaia, Oleg Shupliakov, Robert M. Cassidy, Olle Lindvall and Jonas Frisén Supplementary Figures

Supplementary Figure 1. Viral vectors and strategy for genetic manipulation of ependymal cells (a) Construction of the lentiviral and adenoviral vectors used for ependymal cell-specific expression and recombination. Both vectors carry an internal cassette for Cre recombinase (Cre) driven by the murine Cytomegalovirus immediate early gene promoter (CMV) in the adenovirus, and by the FoxJ1 promoter in the lentivirus. The self-inactivating lentiviral transfer vector is shown in the proviral form: LTR regions (U3, R, U5) with a deletion of 400bp including the enhancer and promoter from U3; major splice donor site (SD); encapsidation signal (ψ) including the 5’ portion of the gag gene (GA); Rev-response element (RRE); splice acceptor sites (SA); the post-transcriptional regulatory element of woodchuck hepatitis virus (WPRE); the central polypurine tract (cPPT) and polyadenylation site A from the Simian Virus 40 (pA). (b) Illustration of the ependyma-specific genetic recombination of the RBP-J and R26R loci in RBP-Jf/f mice. After Cre recombination exon 6 and 7 in the RBP-J locus are removed and RBP-J expression inhibited. Cre recombination also induces expression of the reporter gene LacZ from the R26R locus in the same cells. Intraventricular injection of a lentivirus expressing Cre under the ependyma-specific promoter FoxJ1 [1] ensures that recombination [2], followed by inhibited Notch signaling and reporter gene expression, only takes place in ependymal cells (c). Nature Neuroscience: doi:10.1038/nn.2268

Nature Neuroscience: doi:10.1038/nn.2268

Supplementary Figure 2. Genetic recombination in ependymal cells (a-d) Intraventricular injection of adenovirus expressing Cre recombinase in Z/EG reporter mice results in specific transduction and recombination (visualized by EGFP) in S100ß-immunoreactive ependymal cells. (e) Cre expression and recombination is limited to multiciliated Crocc-immunoreactive ependymal cells. Crocc (also known as Rootletin) is a marker for cells with motile cilia. (f) Cre expression or recombination is never seen in Gfap-immunoreactive astrocytes. (g-h) Electron microscopy localizes Cre-immunoreactivity exclusively to multiciliated ependymal cells. The boxed area in the light micrograph in (g) is shown in the electron micrograph of the adjacent ultrathin section in (h). Non-ependymal ventricle-contacting cells were invariably unlabeled (n=573 cells). Two days after injection of Adeno Cre more than 90 % of the ependymal cells in the lateral ventricle wall expressed Cre. Out of 1570 Cre expressing cells analyzed with confocal microscopy, none was located in the subventricular zone, and all except two cells (0.0013 %) expressed S100ß and had ependymal cell morphology. The two non-ependymal cells were small, round and located supraependymally, i.e. in the lumen of the ventricle and appeared to be leukocytes (data not shown). Injection of FoxJ1-Cre lentivirus into the lateral ventricle of adult mice resulted in Cre expression in more than 80% of ependymal cells, and recombination (assessed by ßgal expression in R26R reporte mice) in almost all transduced cells. (i-l) Lentiviral expression of EGFP/Cre from the FoxJ1 promoter is restricted to S100ß-immunoreactive ependymal cells. EGFP/Cre expression is restricted to Crocc-immunoreactive multiciliated ependymal cells (m) and is never seen in Gfap-immunoreactive astrocytes (n). A subset of subventricular zone astrocytes contacts the ventricle and have an immotile primary cilium1. FoxJ1 is not expressed in cells with primary cilia but is restricted to cells with motile cilia2,3 . (o-p) Ultrastructural analysis localizes Cre-immunoreactivity strictly to multiciliated ependymal cells. The boxed area in (o) is shown in the electron micrograph of the adjacent ultrathin section in (p). Arrowheads indicate osmium labeled lipid droplets. The orientation of all sections in this and subsequent supplemental figures is the same and indicated by arrows in (a). d, dorsal; m, medial. Nuclei are visualized with DAPI in all merged images and appear blue. All analyses of virus-injected brains were conducted in the hemisphere contralateral to the injection. Scale bars: 10 µm in (a) and 5 µm in (h and p). The two different strategies for genetic recombination share a strict specificity for ependymal cells, but differ with regard to kinetics and efficiency. Adenoviral expression of Cre resulted in expression of recombination reporter alleles within two days, whereas recombination was slower after lentiviral injection. We did not detect any signs of toxicity after adenoviral injection. However, dying ependymal cells and a gradual loss of ependymal cells started four weeks after injection of the FoxJ1-Cre lentivirus, for which reason we have limited our analyses to within four weeks after lentiviral injection. In this type of approach the interpretation rests on the specificity of the targeting of recombination. It is difficult to formally exclude the possibility that there may be a minute number of undesired cells that may be labeled, below the level of detection by rigorous analysis, which could affect the interpretation of the results. There are several reasons why labeling of even extremely few non-ependymal cells is unlikely in our study. First, intraventricular injections of adeno- and lentivirus have previously been reported by

Nature Neuroscience: doi:10.1038/nn.2268

several independent groups to specifically transduce ependymal cells (see main text for references). The use of an ependymal specific promoter (FoxJ1) for lentiviral expression of Cre further guarantees the specificity in our study. The specificity of adenoviral transduction appears to be due to the specific expression of the adenovirus receptor CAR/CVADR in ependymal cells in the adult lateral ventricle wall4. It is possible to transduce non-ependymal cell types in the ventricle wall, but that requires orders of magnitude higher titers (see for example5). Second, the expression of Cre under the ependymal specific FoxJ1 promoter allowed us to study whether recombined cells that appeared in the subventricular zone aberrantly expressed Cre. This was never the case. All recombined cells appearing in the subventricular zone in RBP-Jf/f mice lacked Cre-immunoreactivity (Fig. 4d-g, Supplementary Fig. 8), demonstrating that these cells derived from cells that used to have an active FoxJ1 promoter and ependymal character. Third, in mice heterozygous for the conditional RBP-J allele, a minute number of recombined cells were seen in the olfactory bulb and, importantly, only after eight weeks. This is not what is expected if there was any transduction of progenitors or neuroblasts in the subventricular zone, as direct labeling of such cells results in large numbers of recombined cells in the olfactory bulb within a week. Fourth, the loss of recombined ependymal cells after ablation of RBP-J confirms that RBP-J is critically important for the maintenance of the ependymal population (Fig. 5b-e). Fifth, ablation of RBP-J results in a robust phenotype with numerous recombined cells delaminating to the subventricular zone, which is not compatible with recombined cells deriving from an extremely small hypothetical non-ependymal population.

Nature Neuroscience: doi:10.1038/nn.2268

Nature Neuroscience: doi:10.1038/nn.2268

Supplementary Figure 3. Additional examples of ependymal cell generated neuroblasts and astrocytes after stroke Recombined cells leave the ependymal layer in response to stroke and differentiate into Gfap+ astrocytes (a-g) and Dcx+ neuroblasts (h-i). (a-d) 72 h after MCAO some recombined cells extend long Gfap immunoreactive processes (a-b, arrowhead, (b) shows part of (a) in higher magnification). (e-f) Two weeks after MCAO recombined cells with a process and Gfap expressioncan still be found (arrowheads). (f) 3D reconstruction of a confocal z-stack of 1 µm thick sections of the cell in (e), showing colocalization of ßgal and Gfap. (g) Six weeks after MCAO a recombined cell is found in the suvcentricular zone with astrocytic morphology and Gfap expression (arrowhead). The hatched line delineates the lateral ventricle wall. (h-k) An ependyma-derived neuroblast (Dcx+) found in the subventricular zone two weeks after MCAO. (h) 1 µm section of a confocal z-stack of the cell, showing colocalization of ßgal and Dcx. (i) 3D reconstruction of the section in (h). (j-k) The next adjacent 1 µm section of the same cells as in (h-i). (k) 3D reconstruction of the section in (j). Scale bars: (a, c) 50 µm, (e, g, k) 25 µm and (f) 10 µm.

Nature Neuroscience: doi:10.1038/nn.2268

Nature Neuroscience: doi:10.1038/nn.2268

Supplementary Figure 4. Functional assessment of the presence of Cre-expressing lentivirus at different timepoints after intraventricular injection Intraventricular injection of FoxJ1-Cre lentivirus in adult R26R reporter mice results in recombination and ßgal expression in S100ß immunoreactive ependymal cells in the third ventricle (a) as well as in the lateral ventricle (b). Cerebrospinal fluid (2-4 µl) aspirated from the contralateral ventricle two min after an intraventricular injection was injected into another adult R26R reporter mouse, resulting in recombined S100ß immunoreactive ependymal cells in the third (c) and lateral (d) ventricle of the recipient mouse five days later (n=3). In contrast, not a single recombined ependymal cell is found in the third (e) or lateral (f) ventricle of animals receiving cerebrospinal fluid aspirated seven days (timepoint corresponding to stroke induction) after injection into the first mouse (n=3). This provides functional evidence that there is no active virus present in the cerebrospinal fluid at the time of stroke and that all ependymal recombination takes place well before stroke is induced. Scale bars: (a, b, c, d) 25 µm and (e-f) 100 µm.

Nature Neuroscience: doi:10.1038/nn.2268

Nature Neuroscience: doi:10.1038/nn.2268

Supplementary Figure 5. Stroke-induced generation of neuroblasts and astrocytes from ependymal cells after temporally controlled genetic recombination (a-c) Illustration of experimental setup. A plasmid expressing CreER from the ependymal cell specific promoter FoxJ1 was electroporated into the lateral ventricle wall of R26R reporter mice and recombination was induced by five daily tamoxifen injections three days later. Three weeks after ended tamoxifen treatment MCAO was performed. (d) Two weeks after stroke, some recombined ependymal cells are still expressing GFP from the electroporated plasmid (empty arrowheads). Another recombined cell, with typical neuroblast morphology, have left the ependymal layer (white arrowhead) and is no longer GFP+, indicating that the ependymal promoter FoxJ1 is no longer active. (e) As seen after virally induced recombination of ependymal cells, ependymal cells labeled by tamoxifen induced recombination respond to stroke by differentiating into astrocytes with a radial process, or into Dcx immunoreactive neuroblasts (f). (f) Co-expression (yellow) of Dcx and ßgal in four consecutive 1 µm thick sections of an ependyma-derived neuroblast. Nuclei are visualized with DAPI (f) and appear blue. Scale bars: (d) 25 µm and (e-f) 10 µm.

Nature Neuroscience: doi:10.1038/nn.2268

Nature Neuroscience: doi:10.1038/nn.2268

Supplementary Figure 6. Notch signaling is reduced in ependymal cells after stroke A reporter assay for detection of canonical Notch signaling in ependymal cells in normal and stroke brain. (a-d) Electroporation of a plasmid containing 12 RBP-J response elements controlling expression of destabilized GFP into the adult lateral ventricle wall reveals active Notch signaling in S100ß immunoreactive ependymal cells in the uninjured brain. For visualization of transfected cells independently of Notch signaling activity a CMV-LacZ plasmid was co-electroporated. More than 99% of cells are transfected by two plasmids when co-electroporated 6. When animals are subjected to MCAO one week after plasmid transfection, a significant decrease in the number of ependymal cells with high Notch signaling activity, and a large increase in the number of ependymal cells displaying no Notch activity (e-h and Fig. 4a) are seen (2-3 days after stroke). Scale bar: 25 µm.

Nature Neuroscience: doi:10.1038/nn.2268

Nature Neuroscience: doi:10.1038/nn.2268

Supplementary Figure 7. Cre mediated recombination results in loss of RBP-J protein in RBP-Jf/f mice (a-c) RBP-J-immunoreactivity is stronger in the walls of the lateral ventricles (LV) and third ventricle (3V) than in the surrounding tissue. All ependymal cells as well as many cells in the subventricular zone and rostral migratory stream (RMS) are strongly RBP-J-immunoreactive. (d-f) Cre mediated recombination, visualized by ßgal expression, results in loss of RBP-J-immunoreactivity three weeks after injection of Lenti FoxJ1-Cre in RBP-Jf/f mice. Note that adjacent ependymal and subventricular zone cells that do not express ßgal, are RBP-J-immunoreactive. Scale bars: 100 µm in (a and c) and 25 µm in (b and d).

Nature Neuroscience: doi:10.1038/nn.2268

Nature Neuroscience: doi:10.1038/nn.2268

Supplementary Figure 8. Neuroblasts derived from ependymal cells Recombined cells still remaining in the ependymal layer of RBP-Jf/f mice three weeks after injection of Lenti FoxJ1-Cre are Cre-immunoreactive and do not express the neuroblast marker Dcx (empty arrowheads in (a-e)), demonstrating that they retain an active FoxJ1 promoter. (b-e) show part of (a) in higher magnification. ßgal+ cells that leave the ependymal layer and express the neuroblast marker doublecortin (Dcx) are not Cre-immunoreactive (arrowhead in (f-l)), demonstrating that the recombination event took place in an ependymal cell that has taken on neuroblast fate. (i-l) show part of (h) in higher magnification. The images in (f-l) show a ventral part of the lateral ventricle where the ependymal layers from the lateral and medial walls meet. Nuclei are visualized with DAPI (a and h) and appear blue. Scale bars: 25 µm in (a, e and h) and 10 µm in (i).

Nature Neuroscience: doi:10.1038/nn.2268

Nature Neuroscience: doi:10.1038/nn.2268

Supplementary Figure 9. Ependymal cell-derived non-neuronal cells after inhibited Notch signaling (a-c) The absolute majority of recombined cells found outside the ependymal layer in RBP-Jf/f mice take on neuronal fate (one ßIII-tubulin-immunoreactive cell is indicated with a white arrowhead), but a few do not display neuronal markers (empty arrowhead). (d-f) A small subset of ependymal cell-derived recombined cells is immunoreactive for the astrocytic marker Gfap (empty arrowhead). Nuclei are visualized with DAPI (a and d) and appear blue. Scale bars: 10 µm in (a) and 5 µm in (d).

Nature Neuroscience: doi:10.1038/nn.2268

Nature Neuroscience: doi:10.1038/nn.2268

Supplementary Figure 10. Neuronal differentiation in the lateral ventricle wall (a) A single recombined ßgal-immunoreactive cell (empty arrowhead) remains in a section from an RBP-Jf/f mouse lateral ventricle wall eight weeks after injection of Adeno Cre. (b-d) This cell is immunoreactive to the mature neuronal marker NeuN, whereas many neighboring cells are not (one example is indicated by white arrowhead). (e and f) show three-dimensional reconstructions of the ßgal+ / NeuN+ cell. Nuclei are visualized with DAPI (a-b) and appear blue. Scale bars: 50 µm in (a), 10 µm in (b) and 5 µm in (e).

Nature Neuroscience: doi:10.1038/nn.2268

Nature Neuroscience: doi:10.1038/nn.2268

Supplementary Figure 11. Ependyma-derived olfactory bulb neurons in RBPf/f mice mature and localize indistinguishably from olfactory bulb neurons normally born in the adult (a) With increasing time after Cre expression, recombined ependyma-derived neurons in the olfactory bulb of RBP-Jf/f mice acquire complex morphology and express the mature neuronal marker NeuN. (a-b) At eight weeks, recombined cells are identifiable as granule cells at the final stages of maturation (class 4 and class 5 cells7) with NeuN-immunoreactive cell bodies located in the granule cell layer and long processes extending into the external plexiform layer (EPL). The class 4 cells (a-b) have elaborate branched apical dendrites, but few spines, in the EPL. (c) Neurons at the last stage of maturation (class 5 cells) have spines (gemmules) at high density on the apical dendrites. Ependyma-derived neurons in the olfactory bulb of RBPf/f mice (a-c) mature and localize indistinguishably from olfactory bulb neurons normally born in the adult as seen in Nestin-CreER x R26R reporter mice (d-f). Scale bars: 25 µm in (a, d), 10 µm in (b, e) and 5 µm in (c, f). References 1. Doetsch, F., Garcia-Verdugo, J.M. & Alvarez-Buylla, A. Regeneration of a

germinal layer in the adult mammalian brain. Proc Natl Acad Sci U S A 96, 11619-24 (1999).

2. Lim, L., Zhou, H. & Costa, R.H. The winged helix transcription factor HFH-4 is expressed during choroid plexus epithelial development in the mouse embryo. Proc Natl Acad Sci U S A 94, 3094-9 (1997).

3. Blatt, E.N., Yan, X.H., Wuerffel, M.K., Hamilos, D.L. & Brody, S.L. Forkhead transcription factor HFH-4 expression is temporally related to ciliogenesis. Am J Respir Cell Mol Biol 21, 168-76 (1999).

4. Johansson, C.B. et al. Identification of a neural stem cell in the adult mammalian central nervous system. Cell 96, 25-34 (1999).

5. Mirzadeh, Z., Merkle, F.T., Soriano-Navarro, M., Garcia-Verdugo, J.M. & Alvarez-Buylla, A. Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain. Cell Stem Cell 3, 265-78 (2008).

6. Barnabe-Heider, F. et al. Genetic manipulation of adult mouse neurogenic niches by in vivo electroporation. Nat Methods 5, 189-96 (2008).

7. Petreanu, L. & Alvarez-Buylla, A. Maturation and death of adult-born olfactory bulb granule neurons: role of olfaction. J Neurosci 22, 6106-13 (2002).

Nature Neuroscience: doi:10.1038/nn.2268