Betacellulin promotes cell proliferation in the neural stem cell niche and stimulates neurogenesis

María Victoria Gómez-Gaviro1,2, Charlotte E. Scott, Abdul K. Sesay, Ander Matheu, Sarah Booth, Christophe Galichet, and Robin Lovell-Badge2

Division of Stem Cell Biology and Developmental Genetics, MRC National Institute for Medical Research, London NW7 1AA, United Kingdom

Edited by Fred H. Gage, Salk Institute, San Diego, CA, and approved December 14, 2011 (received for review November 10, 2010)

Neural stem cells (NSCs) reside in specialized niches in the adult further up-regulated by SDF-1, suggesting a role for the EGF mammalian brain, including the subventricular zone and the family of growth factors in the regulation of NSC function (4, 6). dentate gyrus, which act to control NSC behavior. Among other Here, we investigate communication between ECs and NSCs cell types within these niches, NSCs are found in close proximity to and unveil that betacellulin (BTC), an EGF-like initially blood vessels. We carried out an analysis of the interaction described to mediate the generation of β cells in the pancreas between endothelial cells and NSCs, and show that betacellulin (9), induces NSC proliferation and enhances neurogenesis. (BTC), a member of the EGF family and one of several signaling Results molecules made by the former, induces NSC proliferation and prevents spontaneous differentiation in culture. When infused Global Analysis of the Effect of ECs on NSCs. In agreement with others, we found that NSCs are located in close proximity (<15 into the lateral ventricle, BTC induces expansion of NSCs and μ SI Results neuroblasts, and promotes neurogenesis in the olfactory bulb and m) to ECs of the microvasculature (Fig. S1 and ). To dentate gyrus, whereas specific blocking antibodies reduce the determine the molecular processes that mediate the effect of number of stem/progenitor cells. BTC-null mice are less able to ECs on NSCs, we cocultured both cell types in a Transwell device (Costar) for 7 d, allowing exchange of soluble factors between regenerate neuroblast numbers compared with WT littermates A β ECs and NSCs but not direct cell-cell contact (Fig. S2 ). NSCs following depletion of proliferating cells using cytosine- -D-arabi- grown in the absence of ECs were used as a negative control. To nofuranoside. BTC acts via both the EGF receptor, located on NSCs, take full advantage of the resources available, we made use of and ErbB4, located on neuroblasts, with the latter explaining why the human NSC line CTX0E03. Using an antibody array, we its effects are distinct from those of EGF itself. Our results suggest observed that a number of receptors presented that BTC could be a good candidate to aid regenerative therapies. differential phosphorylation in the presence of ECs, including the EGF receptors ErbB1 and ErbB4; the FGF receptors 3 and regeneration | vasculature | choroid plexus | cerebral spinal fluid | 4; the receptors for A3, A4, and B6; the VEGF receptors neurospheres 1 and 2; and the glial-derived neurotrophic factor receptor c-Ret (Fig. S2B and Dataset S1). Antibody array analysis of soluble factors revealed a number eural stem cells (NSCs) reside in a specialized microenvi- NEUROSCIENCE Nronment, known as a stem cell niche, that shapes their fate of chemokines and cytokines increased in the coculture super- (1, 2). Two main NSC niches have been described in the adult natant compared with either NSCs or ECs alone, as well as mouse brain: the subventricular zone (SVZ) and the dentate growth factors of the hepatocyte [HGF and mac- gyrus (DG) in the hippocampus (1, 2). Located on the lateral rophage stimulating protein alpha (MSPa)] and EGF (BTC) C wall of the lateral ventricles, the SVZ is composed of different families (Fig. S2 and Dataset S2). To investigate the effect that cell populations, including a monolayer of ependymal cells that EC-released factors have on NSCs, we extracted RNA from lines the ventricle, NSCs, transit-amplifying cells, neural pro- NSCs, either grown alone or in coculture with ECs, and analyzed expression profiles using oligonucleotide DNA microarrays. genitors (neuroblasts), astrocytes, and a dense network of blood > vessels. NSCs extend a process to the ventricle through the NSCs grown in the presence of ECs showed an increase 1.4- ependymal layer and also to endothelial cells (ECs) in the niche, fold in 679 , compared with NSCs grown alone, and a de- crease in 1,898 genes (Datasets S3 and S4). and therefore maintain contact with both the ventricular space Methods and the blood vessels (3). analysis ( ) suggested that coculture with ECs stimulates In the SVZ, which is densely vascularized, neurogenesis is ribosomal RNA and protein synthesis in NSCs, whereas it represses the regulation of gene transcription and cell signaling, associated with the vasculature and most proliferating cells are neurogenesis, cell growth, the Wnt pathway, and the regulation found adjacent or very close to blood vessels (4, 5). GFAP+ of the cell cycle (Datasets S5–S10). Validation of these results astrocytic stem cells and transit-amplifying progenitor cells are by quantitative reverse-transcribed PCR (qRT-PCR) confirmed located in the proximity of the capillaries and chains of neuro- reduced expression of the negative regulators of the cell cycle blasts line the vessels as they migrate along the rostral migratory CDKN1A/p21 and CDKN1C/p57; their regulator FOXO3A; and stream (RMS), although without direct contact (4, 5). Progenitor the differentiation markers GFAP, MAP2, and OLIG2 in cells are recruited to the niche vasculature by SDF-1, and their continuing interaction is dependent on α6β1 (5, 6). The functional effect that ECs exert on NSCs can be reproduced Author contributions: M.V.G.-G., C.E.S., and R.L.-B. designed research; M.V.G.-G., C.E.S., A.K.S., in vitro. ECs, but not smooth muscle cells, induce NSC pro- A.M., S.B., and C.G. performed research; M.V.G.-G. contributed new reagents/analytic liferation in coculture experiments, prevent their differentiation, tools; M.V.G.-G., C.E.S., and R.L.-B. analyzed data; and M.V.G.-G. and R.L.-B. wrote and enhance their neurogenic capacity (7). Both ECs and the paper. ependymal cells support NSC self-renewal, at least in part, by The authors declare no conflict of interest. secreting pigment epithelium-derived factor (8). However, al- This article is a PNAS Direct Submission. though these data strongly suggest that blood vessels have an 1Present address: Servicio de Cardiología, Hospital General Universitario Gregorio Marañón, impact on NSCs, niche homeostasis, and neurogenesis, the un- 28007 Madrid, Spain. derlying molecular mechanisms are far from being understood 2To whom correspondence may be addressed. E-mail: [email protected] or rlovell@ and are likely to involve additional components. Interestingly, nimr.mrc.ac.uk. stem/progenitor cells localized around capillaries in the SVZ This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. show strong expression of EGF receptor (EGFR), which is 1073/pnas.1016199109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1016199109 PNAS | January 24, 2012 | vol. 109 | no. 4 | 1317–1322 Downloaded by guest on September 26, 2021 the presence of ECs (Fig. S2D), suggesting that EC-derived induce neurosphere growth (Fig. 1 B and C) and to derive and factors promote NSC proliferation while inhibiting spontaneous grow secondary neurospheres (Fig. 1D), indicating that BTC differentiation. induces NSC self-renewal. In combination with the prosurvival To determine whether some of the identified in the factor FGF, which is generally used for the growth of neuro- analysis of the coculture supernatant were sufficient to stimulate spheres with EGF, BTC showed similar efficiency to EGF at 20 NSC growth, we studied the ability of NSCs to form neuro- ng/mL and higher efficiency at lower concentrations (Fig. 1E). In spheres in the presence of HGF, MSP, BTC, Gro, Serpin, or addition, whereas a small percentage of spontaneously differ- soluble tumor necrosis factor-receptor II. As shown in Fig. S2E, entiating neurospheres could be detected in the presence of EGF both HGF and BTC increased the number of neurospheres in (20 ng/mL), BTC reproducibly blocked spontaneous differenti- culture. To test the functional relevance of the increased phos- ation (Fig. S3A). Exposure of NSCs to BTC plus FGF also phorylation of VEGF receptors 1 and 2 in the presence of ECs, mimicked the effect of ECs by reducing the expression of dif- we cultured neurospheres with VEGF-A in combination with ferentiation markers and cell cycle regulators (Fig. S3B), com- FGF and/or EGF. VEGF-A synergized with FGF to increase the pared with FGF plus EGF, reinforcing the idea that BTC was number of neurospheres, although it was not able to increase the more efficient than EGF. Immunofluorescence analysis of NSCs number of neurospheres further in the presence of FGF + EGF grown in the presence of BTC (20 ng/mL) for 10 d and then as shown by both BTC and HGF (Fig. S2F). Moreover, VEGF-A allowed to differentiate in the absence of BTC showed that BTC- addition produced smaller neurospheres compared with FGF stimulated NSCs were able to differentiate into the three differ- and EGF (Fig. S2G). VEGF-C acting via VEGF receptor 3 (both ent lineages: neurons (Tuj1), oligodendroglia [2',3'-cyclic nucle- absent from the antibody arrays used here) has recently been otide 3'-phosphodiesterase (CNPase)], and astrocytes (GFAP), shown to stimulate NSC self-renewal and thereby promote maintaining their full differentiation potential (Fig. S3C). (nonspecifically) both neurogenesis and gliogenesis (10). BTC Is Expressed by ECs and the Choroid Plexus. To identify the cell BTC Stimulates Neurosphere Growth. Because it has already been type producing BTC in the coculture, we analyzed RNA from described that HGF can stimulate NSC proliferation (11), and NSCs and ECs by qRT-PCR. We found that ECs were expressing given the relatively weak effects of VEGF-A, we focused on the BTC mRNA, whereas it was barely detectable in NSCs, re- potential of BTC as an NSC growth factor. We investigated gardless of whether they were cocultured or grown alone (Fig. whether BTC was necessary for the action of ECs on NSCs by S4A). In addition, we observed that the EC lines ccec and adding a neutralizing anti-BTC or an isotype control antibody to b.End.3 expressed BTC protein at levels comparable to the the coculture. As shown in Fig. 1A, blockade of BTC decreased pancreatic cell line PARC1 (Fig. S4B). Analysis of BTC ex- the number of neurospheres induced by EC supernatant. We pression in the adult brain showed the highest BTC expression in also compared the efficiency of BTC with that of EGF by the cerebellum and lower amounts in the cortex and the SVZ growing mouse NSCs in the presence of these factors, either and DG NSC niche regions (Fig. S4D). Immunofluorescence combined or separately. We found that BTC is sufficient to analysis of BTC distribution in the brain revealed BTC staining

Fig. 1. BTC stimulates neurosphere growth. (A) Adult mouse NSCs were grown in Transwell devices in the presence of ECs and were treated with 10 μg/mL anti-BTC blocking antibody or an isotype control. Neurosphere number is expressed as the average per well ± SE. (B and C)NSCswerestimulatedasin- dicated with 20 ng/mL FGF, EGF, and/or BTC. Neurosphere number (B) and diameter (C) were determined after 10 d and expressed as the average ± SE. *P < 0.05; **P < 0.005; and ***P < 0.0005 vs. FGF or as indicated (one-way ANOVA plus Bonferroni posttest). (D) Primary neurospheres were generated in the presence of BTC (20 ng/mL) for 10 d, after which they were disgregated, seeded at the initial concentration, and grown with BTC to generate secondary neurospheres. 1ry,primary;2ry, secondary. (E)NSCsweregrownasinB in the presence of FGF (20 ng/mL) plus the indicated concentrations of EGF or BTC, and the diameter ± SE was quantified.

1318 | www.pnas.org/cgi/doi/10.1073/pnas.1016199109 Gómez-Gaviro et al. Downloaded by guest on September 26, 2021 within ECs (Fig. S4F), with a similar pattern to that observed for proliferation rather than reduced apoptosis, because we detected other soluble factors (6, 8). Interestingly, BTC mRNA expres- very few apoptotic cells in the SVZ by TUNEL staining (Fig. S5B). sion and protein staining were also observed in the choroid To determine whether endogenous BTC was necessary for plexus of the lateral ventricle (Fig. S4 D and E), which was maintenance of the stem/progenitor population in the SVZ, we stronger than that observed in blood vessels. In agreement with infused blocking anti-BTC antibody or an isotype control anti- these results, we detected BTC in mouse cerebrospinal fluid body into the lateral ventricle and analyzed the SVZ 7 d later. As (CSF) by Western blot analysis (Fig. S4C), suggesting that both shown in Fig. 2C and Fig. S6A, blockade of endogenous BTC blood vessels and the choroid plexus produce and secrete BTC in activity resulted in a decrease in the number of SOX2+ and the brain. Glast+ stem/precursor cells in the SVZ, together with fewer PSA-NCAM+ neuroblasts, suggesting that BTC is both neces- BTC Stimulates Growth of NSCs and Precursor Cells in the Adult sary and sufficient to regulate proliferation of NSCs and neu- Mouse Brain. We performed a series of experiments infusing BTC roblasts. Preincubation with recombinant BTC neutralized directly into the brain lateral ventricle using an osmotic mini- effects of the blocking antibody, demonstrating the specificity of pump that allows continuous delivery of the protein. Brains were B + fl the result (Fig. S6 ). A decrease in proliferating (BrdU ) analyzed after 1, 4, or 7 d by immuno uorescence using anti- SOX2+ cells was also observed in the DG after infusion of anti- bodies directed against different SVZ cell populations. We BTC (Fig. S6E). found that BTC progressively induced proliferation of SOX2+ To find out which cell subpopulation in the SVZ first reacted cells, which more than quadrupled their number after 1 wk (Fig. A B to BTC, we injected BrdU i.p. and infused BTC or vehicle into 2 and ). Accordingly, we found a similar increase in the fi number of cells positive for BrdU, which labels proliferating the lateral ventricle for 24 h. We quanti ed the percentage of cells in each cell population that had duplicated their DNA cells, and a proportional increase in the SVZ thickness. Whereas + C fi cells positive for Glast, a nonexclusive marker of NSCs and some (BrdU ). As shown in Fig. 2 , BTC signi cantly increased the percentage of SOX2+ cells and neuroblasts that were also astrocytes, were also found to be increased in response to BTC, + + + only a modest increase in the number of GFAP+, Nestin+, and BrdU . No increase in total SOX2 or PSA-NCAM cells was Mash1+ cells was observed, and the number of CD133+ epen- detected within the first 24 h (Fig. 2B), suggesting that these cell dymal cells remained unaltered (Fig. 2 A and B and Fig. S5A). A subpopulations enter the cell cycle within the first 24 h but need similar response was detected in the DG, where BTC induced longer to complete cell division. proliferation of SOX2+ cells (Fig. S6C). A progressive increase Together, these results demonstrate that BTC induces ex- in the number of neuroblasts was also observed, as determined pansion of progenitor populations in the mouse brain, notably with an anti–polysialic acid neural cell adhesion molecule (PSA- NSCs and neuroblasts. We found that this effect is mediated by NCAM) antibody, suggesting that BTC could induce neuro- both EGFR and ErbB4, which, in turn, activate the MEK/Erk genesis. These effects were likely attributable to increased and Akt signaling pathways (Fig. 3C, Fig. S7, and SI Results). In NEUROSCIENCE

Fig. 2. BTC stimulates growth of NSCs and precursor cells in the SVZ of the adult mouse brain in vivo. (A and B) Vehicle (Control) or BTC (400 ng/d) was infused into the mouse lateral ventricle (LV), and mice were killed 1, 4, or 7 d later. For BrdU experiments, BrdU (10 μg/g) was administered daily by i.p. injection for 7 d. Brains were fixed, sectioned, and analyzed by immunofluorescence using antibodies directed against Sox2, BrdU, PSA-NCAM, Glast, GFAP, CD133/Prominin, and Nestin (additional examples are provided in Fig. S1). Nuclei were counterstained using DAPI. (Scale bar, 100 μm.) (B) Average number of cells ± SE in each population were quantified in the SVZ. (C) BTC or vehicle (Control) was infused into the lateral ventricle for 24 h, and a single dose of BrdU (10 μg/g) was administered i.p. at the time of cannula implantation. The percentage of proliferating cells was quantified as 100 × the number of cells positive for each cell population marker that was also positive for BrdU. *P < 0.05; **P < 0.005; ***P < 0.0005.

Gómez-Gaviro et al. PNAS | January 24, 2012 | vol. 109 | no. 4 | 1319 Downloaded by guest on September 26, 2021 contrast, EGF mainly activates EGFR and signals through the or vehicle. After 3 d, immunofluorescence analysis showed a MEK/Erk pathway. fourfold increase in the number of SOX2+ cells after BTC treatment (Fig. 4 A and B), together with an increase in the BTC Increases Neurogenesis. To investigate whether BTC induces number of BrdU+ cells, all of which were also positive for SOX2. neurogenesis in vivo, we bred R26REYFP into mice carrying Because only quiescent NSCs remain after AraC treatment, this Glast::CreERT2. YFP expression was activated in adult Glast+ also confirms that BTC can act on these cells. To determine progenitor cells by tamoxifen injection for 5 d. BTC or vehicle whether BTC was necessary for replenishment of the SVZ niche, was then infused into the lateral ventricle for 7 d. Ten days after we treated WT and BTC-null mice with AraC for 6 d and removing the minipump, a clear increase in Glast+ cell-derived allowed regeneration to proceed for a further 3 or 6 d. Com- YFP+ neurons was detected in the olfactory bulb of mice infused pared with WT, BTC-null animals showed reduced levels of with BTC compared with vehicle (Fig. 3 A and B and Fig. S8). SOX2+ NSCs and proliferating cells after 3 d (Fig. 4D) and far Similar data were obtained with a method (12) whereby NSCs fewer PSA-NCAM+ neuroblasts after 6 d (Fig. 4 C and D). are labeled after a single injection of an adenovirus Cre into the These results suggest that BTC plays a key role in the timely lateral ventricle of R26REYFP mice (Fig. S8). These results regeneration of the NSC niche. suggest that BTC induces expansion of NSCs and neuroblasts in the SVZ and that these cells subsequently migrate to the olfac- Discussion tory bulb to contribute to the neuron population. Similarly, we The molecular mechanisms underlying the effects of ECs on observed increased neurogenesis in the DG using a comple- NSCs are largely unknown. Here, we have identified BTC as an mentary approach. We infused BTC into the lateral ventricle for EC-derived factor capable of inducing NSC proliferation and 7 d, injected BrdU i.p. daily during the same period, and pro- neurogenesis. BTC belongs to the EGF family of growth factors, cessed the brains after 10 d. Immunofluorescence analysis of which bind receptors of the ErbB family and induce their di- BrdU and the neuron markers NeuN and Tuj1 showed an in- merization (13). Interestingly, stem and progenitor cells localized crease in the number of BrdU+ neurons in the DG 10 d after around capillaries in the SVZ niche show strong expression of implantation of the minipump (Fig. S9), confirming that BTC EGFR (4). The effect of different ErbB ligands on the SVZ enhances neurogenesis. appears to be unique. Infusion of EGF into the lateral ventricle induces disorganized proliferation and invasion of progenitors BTC Mediates Regeneration of the NSC Niche. We next analyzed the (transit-amplifying cells), as well as formation of oligodendrog- capacity of BTC to induce regeneration of the SVZ. We ad- lial cells, although decreasing neuroblast differentiation and ministered the antimitotic drug cytosine-β-D-arabinofuranoside neurogenesis (14–18). This is in clear contrast to the effects of (AraC) directly into the lateral ventricle using osmotic mini- BTC, which induces organized expansion of NSCs, some transit- pumps. Six days later, we implanted minipumps delivering BTC amplifying cells, and particularly neuroblasts in the SVZ and

Fig. 3. BTC induces neurogenesis in the olfactory bulb. (A and B) R26REYFP mice were crossed to Glast::Cre-ERT2 mice, and YFP expression was induced in Glast+ adult progenitors by injecting tamoxifen for 5 d. BTC or vehicle was infused for 7 d, and the presence of Glast+ cell-derived neurons in the olfactory bulb was analyzed 10 d (B) and 21 d (A and B) later. (Scale bar, 50 μm.) The percentage of YFP+ cells that were also NeuN+ was scored in three random fields on view. White arrows indicate YFP/NeuN double positive cells. (C) BTC activates EGFR and ErbB4 in vivo. BTC (400 ng/d) or vehicle was infused into the lateral ventricle (LV), and activation of EGFR and ErbB4 was determined by immunofluorescence using phospho-specific antibodies. (Scale bar, 100 μm.) (Inset) p-ErbB4 staining in the SVZ. (Scale bar, 50 μm.)

1320 | www.pnas.org/cgi/doi/10.1073/pnas.1016199109 Gómez-Gaviro et al. Downloaded by guest on September 26, 2021 Fig. 4. BTC contributes to the regeneration of the NSC niche. (A and B) AraC [2% (wt/vol) in saline] was infused into the lateral ventricle (LV) for 6 d, and either vehicle (AraC + Control) or BTC (AraC + BTC; 400 ng/d) was infused for 3 d. Vehicle alone was used as a negative control for AraC (No AraC). BrdU (10 μg/ g) was administered daily by i.p. injection for the last 7 d. Brains were fixed, sectioned, and analyzed by immunofluorescence using the antibodies indicated

on the left. (Scale bar, 100 μm.) (B) Number of cells was quantified only in the SVZ and expressed as the average ± SE. WT and BTC-null mice were treated with NEUROSCIENCE AraC for 6 d as in A and analyzed 3 d (D) or 6 d (C and D) later. Cells were quantified as in B. (Scale bar, 100 μm.) *P < 0.05; **P < 0.005; and ***P < 0.0005 BTC vs. control (B) or BTC-null vs. WT at each time point (D) as determined by the Student t test. #P < 0.05; ##P < 0.005; and ###P < 0.0005 for AraC vs. no AraC for each time point and condition.

DG, without leading to an invasion of the subjacent tissue. Some helps to maintain the stem/progenitor population in the SVZ; of the differences between BTC and EGF are likely to be at- indeed, choroid plexus-derived BTC may be one of the factors tributable to the more promiscuous activity of BTC, which can contributing to the existence of an NSC niche in the SVZ. function via EGFR and ErbB4, whereas EGF does not bind However, the anti-BTC neutralizing antibody also decreased the ErbB4. Thus, BTC can affect neuroblast proliferation directly, number of proliferating SOX2+ cells in the DG, suggesting that because such cells express ErbB4, in addition to promoting ex- endogenous BTC may be part of the vascular NSC niche. In this pansion of NSCs via EGFR and/or ErbB4. The finding that many regard, we found BTC expression in primary ECs and EC lines in neuroblasts persist as clusters, particularly in the SVZ, might culture, both mRNA and protein, and we observed BTC suggest that BTC action also leads to their increased aggregation immunostaining in brain blood vessels, notably in micro- in a manner similar to that shown for NRG1 (19). However, this capillaries in the SVZ. BTC expression in the latter was strongest may simply reflect the limited size of the pathway normally taken toward the surface of the ECs away from the lumen, suggesting by neuroblasts within the RMS (i.e., with insufficient substrate, that it is secreted into the niche rather than the bloodstream. only a subset of the cells can form migrating chains, whereas the Interestingly, the metalloproteinase responsible for BTC shed- remainder adhere only to each other and remain behind in the ding and release to the extracellular milieu, ADAM10, shows SVZ). Importantly, additional BTC increases the number of a similar distribution to that of BTC, expressed in the choroid neurons derived from SVZ cells in the olfactory bulb and new plexus and in the blood vessels of the developing and adult brain (BrdU+) early neurons in the DG, indicating that BTC induces (20, 21). both NSC proliferation and neurogenesis. This is a critical dif- Clearly, several factors found in NSC niches, and made by ference between EGF and BTC. Further differences are outlined various cell types within the niche or present in CSF, have in vitro, where low concentrations of BTC stimulate neurosphere overlapping roles in the behavior of NSCs and their progeny. In growth and prevent spontaneous differentiation more efficiently contrast to the blocking antibody experiments, where the effect than EGF. This could be partially explained by activation of both on BTC will be acute, Btc-null mutant mice appear grossly the Erk and Akt pathways by BTC, whereas EGF activates only normal. However, when challenged with AraC, the recovery of the former (Fig. S7 and SI Results). the niche was severely compromised. This suggests that BTC may Two main routes are responsible for the delivery of small be especially relevant in situations where repair is needed. In molecules to the brain: the vascular network and the ventricles, summary, our results show that BTC is produced endogenously where CSF in the latter is made largely by the choroid plexus (4). in the brain and that it is sufficient to induce NSC proliferation We found high levels of endogenous BTC in both. Our blocking and neurogenesis. Our findings suggest that BTC has therapeutic antibody experiments imply that ventricular activity of BTC potential for the treatment of neurodegenerative diseases.

Gómez-Gaviro et al. PNAS | January 24, 2012 | vol. 109 | no. 4 | 1321 Downloaded by guest on September 26, 2021 Methods sections were fixed in 8% (wt/vol) PFA and staining was performed without Triton X-100. An extended version of the experimental procedures is provided in SI Methods. BTC Infusion. Infusions were carried out as previously described (15, 19). Briefly, osmotic minipumps (Alzet 1007D; flow rate of 0.5 μL/h) were loaded Cell Culture. NSCs were isolated from the mouse SVZ as previously described with BTC (3.3 μgin100μL PBS, 0.1% BSA) or vehicle. Cannulas were (22). The human NSC line CTX0E03 (Reneuron) (23) was grown in NSC implanted at 0.0 mm relative to bregma, 1.2 mm lateral and 3.5 mm deep, growth medium in the presence of EGF (20 ng/mL) and FGF-2 (20 ng/mL). and the attached minipumps were implanted s.c. in the intercapsular region. Mouse NSCs were grown in NSC growth medium, and the different growth Mice were killed 1 wk after minipump implantation and analyzed as de- factors were added at the indicated concentrations. Human umbilical cord scribed above. For regeneration experiments, Ara-C [2% (wt/vol)] was in- endothelial cells (HUVECs) were grown in M199 supplemented with 10% fused 1 wk before BTC infusion. For fate mapping, conditional R26REYFP (vol/vol) FCS and Endothelial Growth Factor (Upstate). For coculture adult male mice, which express the YFP reporter after Cre-mediated re- experiments, HUVECs were grown in the upper chamber of a Transwell combination, were crossed with Glast::Cre-ERT2 mice, which express a ta- device with a 0.4-μm pore (Costar) until confluence was reached. Neuro- moxifen-inducible form of Cre in Glast-expressing cells. Recombination was spheres were disaggregated and seeded in the bottom chamber in NSC induced by injecting tamoxifen for 5 d, and BTC or vehicle was infused. Cells medium containing no EGF or FGF-2. were quantified as described in SI Methods.

Immunostaining. Adult MF1 mice were perfused intracardially with 4% ACKNOWLEDGMENTS. We are grateful to C. Wise, R. Subramaniam, and (vol/vol) paraformaldehyde (PFA) for fixation. Brains were excised and C. Parras for technical support. Glast::CreERT2 mice were generously mounted in agarose, and 70-μm sections were sliced in the vibratome. Each supplied by Magdalena Goetz. CTX0E03 cells were a gift from Dr. J. Sinden, section was blocked using 10% (vol/vol) donkey serum in PBS/0.1% Triton and mouse CSF was supplied by Dr. E. Bides, Dr. C. Guaza, and Dr. P. Bovolenta. This work was supported by UK Medical Research Council X-100 for 30 min at room temperature and incubated with primary anti- Grant U117512772 and by a Quantum grant from the US National bodies overnight at 4 °C. BrdU immunostaining was performed as described Institutes of Health (National Institute of Biomedical Imaging and Bio- (12). Sections were washed and incubated with secondary antibodies, and engineering) (to R.L.-B.). M.V.G.-G. was supported by a Marie Curie Intra- DAPI was added before mounting. For ICAM2/BTC coimmunostaining, European Fellowship.

1. Moore KA, Lemischka IR (2006) Stem cells and their niches. Science 311:1880– 14. Kuhn HG, Winkler J, Kempermann G, Thal LJ, Gage FH (1997) Epidermal growth 1885. factor and fibroblast growth factor-2 have different effects on neural progenitors in 2. Doetsch F (2003) A niche for adult neural stem cells. Curr Opin Genet Dev 13: the adult rat brain. J Neurosci 17:5820–5829. 543–550. 15. Doetsch F, Petreanu L, Caille I, Garcia-Verdugo JM, Alvarez-Buylla A (2002) EGF 3. Mirzadeh Z, Merkle FT, Soriano-Navarro M, Garcia-Verdugo JM, Alvarez-Buylla A converts transit-amplifying neurogenic precursors in the adult brain into multipotent (2008) Neural stem cells confer unique pinwheel architecture to the ventricular sur- stem cells. Neuron 36:1021–1034. Cell Stem Cell – face in neurogenic regions of the adult brain. 3:265 278. 16. Jin K, et al. (2002) Heparin-binding -like growth factor: Cell 4. Tavazoie M, et al. (2008) A specialized vascular niche for adult neural stem cells. Hypoxia-inducible expression in vitro and stimulation of neurogenesis in vitro and Stem Cell – 3:279 288. in vivo. J Neurosci 22:5365–5373. 5. Shen Q, et al. (2008) Adult SVZ stem cells lie in a vascular niche: A quantitative analysis 17. Gonzalez-Perez O, Romero-Rodriguez R, Soriano-Navarro M, Garcia-Verdugo JM, of niche cell-cell interactions. Cell Stem Cell 3:289–300. Alvarez-Buylla A (2009) Epidermal growth factor induces the progeny of sub- 6. Kokovay E, et al. (2010) Adult SVZ lineage cells home to and leave the vascular niche ventricular zone type B cells to migrate and differentiate into oligodendrocytes. Stem via differential responses to SDF1/CXCR4 signaling. Cell Stem Cell 7:163–173. Cells 27:2032–2043. 7. Shen Q, et al. (2004) Endothelial cells stimulate self-renewal and expand neurogenesis 18. Gampe K, Brill MS, Momma S, Götz M, Zimmermann H (2011) EGF induces CREB and of neural stem cells. Science 304:1338–1340. ERK activation at the wall of the mouse lateral ventricles. Brain Res 1376:31–41. 8. Ramírez-Castillejo C, et al. (2006) Pigment epithelium-derived factor is a niche signal 19. Ghashghaei HT, et al. (2006) The role of -ErbB4 interactions on the pro- for neural stem cell renewal. Nat Neurosci 9:331–339. Proc Natl Acad Sci USA 9. Dunbar AJ, Goddard C (2000) Structure-function and biological role of betacellulin. liferation and organization of cells in the subventricular zone. – Int J Biochem Cell Biol 32:805–815. 103:1930 1935. 10. Calvo C-F, et al. (2011) Vascular endothelial 3 directly regu- 20. Sahin U, et al. (2004) Distinct roles for ADAM10 and ADAM17 in ectodomain shedding J Cell Biol – lates murine neurogenesis. Genes Dev 25:831–844. of six EGFR ligands. 164:769 779. fi 11. Kokuzawa J, et al. (2003) promotes proliferation and 21. Lin J, Luo J, Redies C (2008) Differential expression of ve members of the ADAM neuronal differentiation of neural stem cells from mouse embryos. Mol Cell Neurosci family in the developing chicken brain. Neuroscience 157:360–375. 24(1):190–197. 22. Doetsch F, Caillé I, Lim DA, García-Verdugo JM, Alvarez-Buylla A (1999) Subventricular 12. Scott CE, et al. (2010) SOX9 induces and maintains neural stem cells. Nat Neurosci 13: zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97:703–716. 1181–1189. 23. Pollock K, et al. (2006) A conditionally immortal clonal stem cell line from human 13. Linggi B, Carpenter G (2006) ErbB receptors: New insights on mechanisms and bi- cortical neuroepithelium for the treatment of ischemic stroke. Exp Neurol 199(1): ology. Trends Cell Biol 16:649–656. 143–155.

1322 | www.pnas.org/cgi/doi/10.1073/pnas.1016199109 Gómez-Gaviro et al. Downloaded by guest on September 26, 2021