Radial Glia Give Rise to Adult Neural Stem Cells in the Subventricular Zone

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Radial glia give rise to adult neural stem cells in the subventricular zone

Florian T. Merkle*†, Anthony D. Tramontin*†, Jose´ Manuel Garcı´a-Verdugo‡, and Arturo Alvarez-Buylla*§

*Department of Neurological Surgery, Developmental and Stem Cell Biology Program, Box 0525, University of California, San Francisco, CA 94143; and ‡Instituto Cavanilles, Universidad de Valencia, 46100 Valencia, Spain

Communicated by Fernando Nottebohm, The Rockefeller University, Millbrook, NY, October 22, 2004 (received for review September 1, 2004) Neural stem cells with the characteristics of astrocytes persist in the ventricle of postnatal day (P) 0 mice (29). We show that these subventricular zone (SVZ) of the juvenile and adult brain. These radial glial cells give rise to neurons, astrocytes, ependymal cells, cells generate large numbers of new neurons that migrate through and oligodendrocytes. More importantly, we show that these the rostral migratory stream to the olfactory bulb. The develop- neonatal radial glial cells give rise to the SVZ astrocytes that mental origin of adult neural stem cells is not known. Here, we maintain neurogenesis in the adult mammalian brain. This work describe a lox–Cre-based technique to specifically and permanently identifies the neonatal origin of adult SVZ neural stem cells. label a restricted population of striatal radial glia in newborn mice. Within the first few days after labeling, these radial glial cells gave Materials and Methods rise to neurons, oligodendrocytes, and astrocytes, including astro- Labeling of P0 Radial Glia by Striatal Adenovirus (Ad) Injection. All cytes in the SVZ. Remarkably, the rostral migratory stream con- protocols followed the guidelines of the Laboratory Animal tained labeled migratory neuroblasts at all ages examined, includ- Resource Center at the University of California, San Francisco. ing 150-day-old mice. Labeling dividing cells with the S-phase Newborn mice were anesthetized by hypothermia and placed marker BrdUrd showed that new neurons continue to be produced onto the platform of a stereotaxic injection rig where their heads in the adult by precursors ultimately derived from radial glia. were stabilized by a customized head mold. Striatal injections (20 Furthermore, both radial glia in neonates and radial glia-derived nl) of Ad were targeted unilaterally as shown in Fig. 1 G and H. cells in the adult lateral ventricular wall generated self-renewing, We avoided the ventricle, VZ, and SVZ by angling the injection multipotent neurospheres. These results demonstrate that radial needle at 45° and following a lateral approach. After injection, glial cells not only serve as progenitors for many neurons and glial animals were returned to their mother and monitored every 10 cells soon after birth but also give rise to adult SVZ stem cells that min until they resumed nursing. continue to produce neurons throughout adult life. This study identifies and provides a method to genetically modify the lineage Immunohistochemistry. Animals were deeply anesthetized and that links neonatal and adult neural stem cells. killed by transcardial perfusion of saline followed by 4% paraformaldehyde. After 24 h in 4% paraformaldehyde, 50-␮m neurogenesis ͉ oligodendrocytes ͉ subependyma vibratome sections were cut and immunostained with antibodies directed against the RC2 antigen (mouse monoclonal IgM, he mammalian brain retains neural stem cells in the subven- Developmental Studies Hybridoma Bank, Iowa City, IA), GFAP Ttricular zone (SVZ) of the lateral ventricular wall. These cells (mouse monoclonal IgG1, Chemicon; rabbit polyclonal IgG, generate new neurons (1–4) that migrate along the rostral DAKO), EGFP (rabbit polyclonal IgG, Novus Biologicals, Littleton, CO; sheep polyclonal Ig, Biogenesis, Bournemouth, migratory stream (RMS) to the olfactory bulb, where they U.K.), doublecortin (guinea pig polyclonal IgG, Chemicon), differentiate into granular and periglomerular interneurons (5, antineuronal nuclei (NeuN; mouse monoclonal IgG1, Chemi- 6). Although they are actively neurogenic, adult SVZ stem cells con), heat stable antigen (CD24; rat monoclonal IgG, Pharm- express glial fibrillary acidic protein (GFAP) and have the ingen), olig2 [mouse monoclonal IgG, a generous gift from morphology and ultrastructure of astrocytes (7–10). These find- David Rowitch (Harvard Medical School, Boston)], ␤-tubulin ings challenge the classical view that all astrocytes are terminally type III (Tuj1; mouse monoclonal IgG2a, Covance, Princeton), differentiated glial cells belonging to a lineage separate from that and O4 (mouse monoclonal IgG, Chemicon) following standard of neurons (7, 11). However, the developmental origin of these protocols. Staining was visualized with secondary antibodies neurogenic astrocytes remains elusive and controversial. conjugated to 7-amino-4-methylcoumarin-3-acetic acid, Cy3, or Here, we tested the hypothesis that adult SVZ neural stem Alexa Fluor 488, 568, 594, or 647 fluorophores or to biotin cells are derived from radial glia. Radial glia have a long, followed by streptavidin and diaminobenzidine. Omission of the RC2-positive (12) basal process that extends from their cell body primary antibodies eliminated staining at all developmental ages in the ventricular zone (VZ) through the parenchyma toward the examined. Sections were counterstained with DAPI. brain surface (13). Radial glia have anatomical features of cells in the astroglial lineage, including endfeet on blood vessels, Electron Microscopy. Tissue processing for electron microscopy intermediate filaments, and glycogen granules. At the end of after ␤-galactosidase histochemistry was performed as described histogenesis, many radial glia differentiate into parenchymal in ref. 30. Cell counting in the neonatal VZ by using electron astrocytes (14, 15). For these reasons, radial glia classically were microscopy was performed as described in ref. 31. The ultra- viewed as immature glial cells that guide neuronal migration and structural criteria to identify radial glia and adult SVZ cell types function as scaffolding for brain development (16, 17). However, are described in refs. 31 and 32. studies in songbirds (18), where radial glia persist into adult life, and work in the developing rodent brain (19–25) indicate that radial glia also function as neural progenitors. We (11) and Abbreviations: Ad, adenovirus; Ad:Cre, Ad expressing Cre recombinase; Ad:GFP, EGFP- others (26–28) have hypothesized that neural stem cells may be expressing Ad; GFAP, glial fibrillary acidic protein; Pn; postnatal day n; SVZ, subventricular contained within what was classically considered the macroglial zone; VZ, ventricular zone. lineage (i.e., neuroepithelial cells 3 radial glia 3 astrocytes). †F.T.M. and A.D.T. contributed equally to this work. In the present study, we genetically tagged a restricted pop- §To whom correspondence should be addressed. E-mail: [email protected]. ulation of radial glia that persist in the lateral wall of the lateral © 2004 by The National Academy of Sciences of the USA

17528–17532 ͉ PNAS ͉ December 14, 2004 ͉ vol. 101 ͉ no. 50 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0407893101 Downloaded by guest on September 26, 2021 Fig. 1. Radial glia are labeled speciﬁcally in the neonatal mouse brain. (A) RC2ϩ radial glial processes extend from the VZ to the brain surface. (B–F) In the region boxed in A, the disappearance of RC2ϩ radial glia correlates with the appearance of GFAPϩ SVZ astrocytes. (G) Radial glial processes can be infected by an Ad injected into the ventrolateral striatum. (H) This injection results in the labeling of cells at the injection site and in the dorsolateral VZ. (I) VZ cells (box in H) have long radial processes that express RC2 (boxed region) and extend from the VZ through the injection site. (J) These cells have the ultrastructure of radial glia. (J Inset) In the same cells (from P2 R26R mice), X-Gal precipitate (arrowheads) is shown at the light microscope. (K and L) In the early postnatal brain, radial glia retract their processes (arrowheads) as they transform into astrocytes in the striatum (K) and in the SVZ (L). (M–P) Their progeny, however, continue to generate large numbers of neuroblasts in the P90 brain (M) that migrate along the RMS to the olfactory bulb (N) and differentiate into granular (O) and periglomerular (P) cells. Cb, cerebellum; Ctx, cortex; LV, lateral ventricle; Stri, striatum.

Viral Diffusion Assay. To determine whether Ad injected in the grafts, but not grafts from cells labeled locally at the injection ventrolateral striatum could be diffusing through the striatum site, produced all of the cell types seen from the injection alone. and infecting cells at or near the VZ, we injected Ad expressing Cre recombinase (Ad:Cre) into the lateral striatum of wild-type BrdUrd Treatment and Immunohistochemistry. To demonstrate the (CD1) P0 pups along with bromophenol blue to visualize the continual production of neurons in the adult, newborn Z͞EG injection site. At various time points after injection (5 and 20 min mice were injected unilaterally with Ad:Cre into the ventrolat- and 1, 4, 8, and 24 h), we killed the animals and microdissected eral striatum. Thirty days later, BrdUrd was added to their ͞ equally sized explants of the injection site, the ipsilateral ven- drinking water (1 mg ml) for 3 days, following a standard tricular wall, and the striatum halfway between these two sites protocol (6). Immediately thereafter or 15 days later, animals (see Fig. 4A, which is published as supporting information on the were perfused with 4% paraformaldehyde as described above. ␮ PNAS web site). We then cultured these explants for 3 days on Vibratome sections of 50 m were cut, treated for BrdUrd top of monolayers of EGFP Ad:Cre-reporting (Z͞EG strain) staining by using a protocol described in ref. 33, and immuno- SVZ astrocytes (n ϭ 3 per condition). We found EGFPϩ cells in stained for EGFP (rabbit polyclonal IgG, Abcam, Cambridge, U.K.), BrdUrd (rat monoclonal IgG, Oxford Biotechnology, monolayers cultured with the injection site explant (Fig. 4B) but Oxfordshire, U.K.), and GFAP (mouse monoclonal IgG, Chemi- never from monolayers cultured with the ventricular wall ex- con). Staining was revealed by secondary antibodies conjugated plant. Furthermore, the number of cells infected by each explant to Alexa Fluor 488, 594, or 647 fluorophores, respectively. rapidly decreased over time; after4hwecould not detect any Omission of the primary antibodies eliminated staining. Double- infectious particles. labeled cells were identified at the light microscope and confirmed by confocal analysis. Grafting of Radial Glial Cell Bodies. To obtain labeled radial glia, we ͞ injected heterozygous Z EG pups at P0, killed the animals at P3, Clonal Generation of Neurospheres. To determine whether radial and exposed the lateral wall of the lateral ventricle under a glia or their adult progeny could form neurospheres, Z͞EG fluorescent dissecting scope. This procedure allowed us to ϩ animals were injected with Ad:Cre at P0, and the lateral visualize the injection site and a well defined patch of EGFP ventricular wall of P2, P10, and P90 animals was dissected, radial glial cell bodies in the VZ (Fig. 2K). We microdissected the dissociated, and placed in neurosphere medium as described in labeled cells in the VZ (n ϭ 6 grafts) and the injection site (n ϭ ref. 6. After amplification in culture, cells were sorted for EGFP 4 grafts) and stereotactically grafted them into the VZ͞SVZ of by FACS on a MoFlo Cytomation (Freiburg, Germany) cell their wild-type littermates, which we then killed at P30. In a sorter at Ϸ100 cells per sec. Upon sorting, we obtained a bimodal ϩ separate experiment, we also grafted labeled cells from the distribution, with EGFP cell peak Ϸ2 orders of magnitude NEUROSCIENCE injection site (n ϭ 6) into the ventrolateral striatum. Radial glial greater than the peak for EGFPϪ cells. Visual inspection con-

Merkle et al. PNAS ͉ December 14, 2004 ͉ vol. 101 ͉ no. 50 ͉ 17529 Downloaded by guest on September 26, 2021 Fig. 2. Radial glia produce all classes of brain cells. (A–J) Radial glial progeny were identified with the confocal microscope by EGFP expression in Z͞EG mice and with the electron microscope by dark, perinuclear grains of X-Gal precipitate in R26R mice (black arrowheads). The progeny include doublecortinϩ neuroblasts (type A cells) (A and B), NeuNϩ neurons (C), ciliated (arrows) CD24ϩ ependymal cells (E and F), and Olig2ϩ oligodendrocytes whose processes (arrows) intercalate between myelinated axons (G and H). (D, I, and J) Radial glia also produce GFAPϩ SVZ astrocytes or type B cells (I and J; intermediate filaments shown in Inset) and neurogenic transit-amplifying type C cells (D). (K) A whole mount of the lateral ventricular wall reveals the injection site deep in the tissue at a deeper focal plane (yellow arrowheads) and a well defined patch of labeled radial glial cell bodies (arrow). (L) When grafted into a wild-type mouse, radial glial cell bodies (but not cell bodies from the injection site) produce all cell types obtained by viral injection alone, including neuroblasts (arrow) and olfactory bulb neurons (yellow arrowhead) in the adult brain. GCL, granule cell layer.

firmed strong EGFP fluorescence in Ͼ98% of sorted cells. We border to the piriform cortex (Fig. 1G). Two days after infection, sorted the cells a second time by using the same parameters to we observed labeling at the injection site and in a restricted achieve an Ϸ99.9% pure population of EGFPϩ cells. Visual region of the dorsolateral striatal VZ, 1.10 Ϯ 0.13 mm (mean Ϯ inspection confirmed that all neurospheres formed by these cells SEM) away from the injection site (Figs. 1H and 2K). Labeled were strongly green fluorescent. Upon plating at clonal density cells in the VZ had the distinctive morphology of radial glia and (20 cells per ␮l), these cells generated neurospheres that were had RC2ϩ processes that traversed the injection site (see below). individually differentiated and stained for EGFP, O4, Tuj1, and Thus, radial glial cell bodies can be labeled retrogradely with Ad. GFAP as described in ref. 6. The rapid infection, short half-life, and limited diffusion of Ad through brain parenchyma (34) result in rapid, localized labeling. Results However, the adenoviral genome is not incorporated into cel- Maturation of the SVZ. We first studied the composition of the lular DNA and becomes diluted with cell division. Therefore, we lateral ventricular wall at different ages. At P0, the striatal VZ used a Cre–lox-based strategy to trace the long-term progeny of contains primarily radial glial cell bodies and a few immature radial glia. We used the same targeting strategy to infect the ependymal cells but no astrocytes (29). The cell bodies of distal processes of radial glia but injected P0 Z͞AP (35), Z͞EG subcortical (striatal) radial glia line the lateral wall of the lateral (36), or R26R (37) mice with Ad:Cre (38). Z͞AP mice express ventricle, and their long RC2ϩ basal processes curve through the alkaline phosphatase (AP), Z͞EG mice express EGFP, and striatum to contact the brain surface (Fig. 1A). To determine the R26R mice express LacZ upon Cre-mediated recombination, so time course of astrocyte formation in the SVZ, we immuno- infected radial glia and their progeny are labeled permanently. stained for GFAP (31) and RC2 to label astrocytes and radial As with Ad:GFP-injected mice, Ad:Cre-injected Z͞AP, glia, respectively, at P0, P2, P4, P6, P8, P10, and P60 (n ϭ 4 per Z͞EG, or R26R mice had APϩ, EGFPϩ,orLacZϩ cells, age group). The disappearance of radial glia from the lateral respectively, at the injection site and in the VZ. We confirmed ventricular wall correlates with the appearance of GFAPϩ that all labeled cells in the VZ were radial glia by immunostain- astrocytes (at P6) directly below the VZ in the developing SVZ ing for RC2 (Fig. 1I) and with electron microscopy (Fig. 1J; 527 (Fig. 1 B–F). We hypothesized that striatal radial glia in the cells analyzed) 2 days after injection. We never detected labeled neonatal brain give rise to the neurogenic astrocytes found in the cells in the medial ventricular wall or on the contralateral side, adult SVZ. indicating that the Ad:Cre did not leak into the ventricular lumen. Furthermore, cells were not nonspecifically labeled at or Radial Glial Targeting and Lineage Tracing. To test this hypothesis, near the VZ by viral diffusion through the parenchyma, because we developed a technique to trace the progeny of a restricted infective Ad:Cre particles completely disappeared from the population of radial glia. We found that radial glia in P0 mice can injection site after 4 h and were never detected near the VZ. be labeled by a replication incompetent, EGFP-expressing Ad Therefore, all labeled cells in the VZ correspond to radial glia (Ad:GFP). To ensure that we were specifically labeling radial infected at their distal processes. glia and not other cells in the VZ or its vicinity, we infected radial glia at their distal basal processes by stereotactically injecting Radial Glia Produce All Four Major Classes of Brain Cells. We then Ad:GFP (20 nl) into ventrolateral striatum of P0 mice near the examined the progeny of radial glia in Z͞AP, Z͞EG, and R26R

17530 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0407893101 Merkle et al. Downloaded by guest on September 26, 2021 mice that survived 4, 7, 10, 15, 30, 45, 90, or 150 days (n Ն 4 per age group) after Ad:Cre injection. At all time points, we observed labeled cells in the VZ and SVZ, at the injection site in the ventrolateral striatum, and scattered stellate astrocytes in the striatum. We identified radial glial progeny by using Golgi- like AP staining for morphology (see Fig. 5 C–L, which is published as supporting information on the PNAS web site), immunofluorescence for cell-type specific markers, and electron microscopy. At short survivals (P4–P10), we observed radial glia retracting their basal process from the injection site and trans- forming into striatal (Fig. 1K) and SVZ (Fig. 1L) astrocytes. From P4 on, the SVZ and RMS contained many migratory neuroblasts (type A cells) (Figs. 1M and 2 A and B), and from P10 on, the ipsilateral olfactory bulb contained large numbers of labeled granular (Figs. 1 M–O and 2C) and periglomerular (Fig. 1 M, N, and P) neurons. In addition, we identified labeled ependymal cells lining the lateral ventricular wall (Fig. 2 E and F) and oligodendrocytes in the corpus callosum and striatum (Fig. 2 G and H). We provide a more detailed description of the generation of ependymal cells from radial glia in a separate study (39). Importantly, we also observed labeled type B cells, the Fig. 3. Radial glia-derived adult stem cells remain actively neurogenic in astrocytic stem cells of the adult SVZ (7–10) (Fig. 2 I and J), and vivo.(A) Radial glia-derived (EGFPϩ) GFAPϩ astrocytes in the adult (P33) SVZ type C cells, the neurogenic transit-amplifying cells that are are labeled by the S-phase marker BrdUrd. (B) GFAP EGFP double-labeling produced by SVZ astrocytes (7, 31, 40) (Fig. 2D), strongly shown at higher magniﬁcation. (C) We also found many double-labeled suggesting that radial glia give rise to adult neural stem cells. migratory neuroblasts. (D) These adult-born neuroblasts migrate to the olfactory bulb and differentiate into interneurons. Radial glia and their adult ϩ Radial Glia, Not Striatal Cells, Act as Progenitors. To demonstrate progeny are self-renewing and multipotent in vitro.(E) EGFP cells isolated that these cell types are derived exclusively from radial glia, we from the ventricular wall of P2, P10, and P90 Z͞EG mice injected with Ad:Cre in the ventrolateral striatum at P0 clonally produce primary neurospheres. microdissected either labeled radial glia from the VZ (Fig. 2K, ϩ arrow) or labeled cells from the injection site (Fig. 2K, arrow- These EGFP neurospheres were sorted by FACS and grown again at clonal ͞ density for several passages to ensure clonality and self-renewal. (F–H) All cells heads) and stereotactically grafted them into the VZ SVZ of in clonally grown neurospheres are EGFPϩ (F) and differentiate into Tuj1ϩ wild-type mice. The pure population of labeled radial glia neurons (visualized by diaminobenzidine, dark brown spots in G), GFAPϩ produced all cell types we observed in Ad:Cre-injected animals, astrocytes, and O4ϩ oligodendrocytes (H). (I) A higher magniﬁcation of a Tuj1ϩ including SVZ astrocytes, neuroblasts, and olfactory bulb inter- neuron (box in G). (J and K) GFAPϩ processes of astrocytes (J) and an O4ϩ neurons (Fig. 2L). However, cells grafted from the injection site oligodendrocyte (K) (box in H). The differentiated neurosphere shown in F–K into the VZ͞SVZ or into the ventrolateral striatum never was isolated from a P90 mouse, but we obtained similar results from all ages produced ependymal cells, oligodendrocytes, neuroblasts, or we examined, demonstrating that both radial glia and their progeny are olfactory bulb neurons. In a separate experiment, we found that multipotent, self-renewing stem cells. (F–H and I–K share the same scale bar.) radial glia could be labeled by adenoviral injection into the lateral ventricle of P0 Z͞AP mice, demonstrating that they contact the ventricular surface. These labeled cells gave rise to neurons in the adult brain long after radial glia have disappeared. all progeny we observed with injection into the ventrolateral But are these cells truly stem cells? striatum, including types B, C, and A cells and olfactory bulb To address this question, we generated neurospheres from the interneurons (Fig. 5 J–L). Thus, the progenitor cells in this lateral wall of the lateral ventricle of P2, P10, and P90 Z͞EG system are those that have the defining characteristics of striatal animals injected with Ad:Cre in the ventrolateral striatum at P0. radial glia: cell bodies in the VZ that contact the ventricular A neurosphere is a floating cell mass that results from the surface and RC2ϩ processes that project through the striatum. expansion of a single stem cell grown in dissociated culture (2, 43–45). Neurospheres are thought to be formed by stem cells Radial Glia Produce Adult Neural Stem Cells. Types B, C, and A cells because they are self-renewing (neurospheres form from disso- compose the neurogenic lineage of the adult brain (7, 30). Types ciated neurospheres) and multipotent (neurospheres have the C and A cells compose transient populations that disappear ability to differentiate into multiple cell types) (46–49). Imme- when neurogenesis is eliminated by treatment with antimitotic diately after isolation from the ventricular wall, all age groups we agents (7, 41, 42). However, we observe large numbers of these examined generated primary EGFPϩ neurospheres (Fig. 3E) labeled cells in adult (up to P150) mice, long after radial glia have that differentiated into neurons, oligodendrocytes, and astro- disappeared. This observation suggests that labeled radial glia cytes. This result demonstrates that radial glia and their progeny generate the actively neurogenic adult stem cell lineage. To test are multipotent in vitro. To demonstrate self-renewal and to this inference, we injected Z͞EG mice with Ad:Cre in the ϩ ensure a pure population of EGFP cells, we passaged primary ventrolateral striatum at P0 and allowed them to survive for 30 ϩ days. At P30, we added 1 mg͞ml of the S-phase marker BrdUrd neurospheres three or more times and then purified EGFP cells to their drinking water for 3 days, as described in ref. 7. We then by FACS. We then passaged these cells at least three more times, killed these animals at P33 (n ϭ 8) or P45 (n ϭ 9) and sorted them again by FACS, and grew them at clonal density (20 ␮ immunostained for EGFP and BrdUrd. In the brains of the P33 cells per l) for three passages. At each passage, we ensured a mice, we found BrdUrd and EGFP double-labeled astrocytes in single-cell suspension by visual inspection after passage through the SVZ (Fig. 3 A and B) and neuroblasts throughout the RMS a cell strainer. After these passages, we hand-picked individual (Fig. 3C). In P45 mice, most of these double-labeled cells had neurospheres and transferred them to separate wells for differ- migrated to the olfactory bulb and matured into interneurons entiation. These clonally generated, radial glia-derived neuro-

(Fig. 3D), but some remained in the SVZ and RMS. Thus, radial spheres from P2, P10, and P90 animals differentiated into NEUROSCIENCE glia generate a population of cells that continue to produce neurons, astrocytes, and oligodendrocytes (Fig. 3 F–K). These

Merkle et al. PNAS ͉ December 14, 2004 ͉ vol. 101 ͉ no. 50 ͉ 17531 Downloaded by guest on September 26, 2021 experiments demonstrate that individual radial glia and their Fundamental questions about neural stem cells could be progeny in the adult brain act as stem cells. addressed by studying their lineage in greater detail. We have demonstrated that individual radial glia and their progeny act as Discussion multipotent stem cells in vitro, but are they multipotent in vivo We demonstrate that radial glia in the neonatal ventricular wall as individual cells or as a population? To what extent does the produce multiple classes of brain cells: astrocytes, oligodendro- potential of radial glia change when they transform into astrocytes, ependymal cells, and neurons. Importantly, we also show cytes? Is this process permanent or reversible? What distin- that these radial glia give rise to adult SVZ stem cells that guishes multipotent radial glia from radial glia that produce only maintain the neurogenic lineage in the adult brain. This con- neurons and astrocytes? To address these questions, the genetic clusion is based on four separate observations. First, infected ϩ recombination technique we developed to label radial glia could radial glia produced GFAP SVZ astrocytes, which have been be adapted to introduce or eliminate a gene of interest and then identified as the neurogenic stem cells in the SVZ (7–10). trace a subset of embryonic or adult stem cells. Such work would Second, the adult RMS was full of labeled migratory neuroblasts shed light on brain development and maintenance and advance that only could have been produced in the adult brain from neurogenic adult stem cells, because radial glia disappear from efforts to manipulate neural stem cells for therapeutic purposes. the brain soon after birth. We confirmed this result by double- labeling SVZ astrocytes, neuroblasts, and olfactory bulb neurons We thank Gail Martin (University of California, San Francisco) for providing Cre reporter mice and Carlos Lois (Massachusetts Institute of with BrdUrd. Finally, we demonstrated that radial glia and their Technology, Cambridge) for providing Ad:Cre; Nuria Flames for help progeny isolated from the P2, P10, and P90 brain clonally with Ad injections and tissue processing; and Cynthia Yaschine and generate self-renewing, multipotent neurospheres. We conclude Matthew G. H. Chun for valuable comments that improved the manu- that striatal radial glia give rise to SVZ astrocytes that continue script. This work was supported by grants from the National Institutes of to generate neurons in the adult brain. This finding extends Health and a generous gift from Frances and John Bowes. F.T.M. was recent evidence that radial glia can generate neurons (18–25) supported by Aging and Neurodegenerative Diseases Training Grant and supports the hypothesis (11, 26–28) that the stem cells of the AG00278-01 from the National Institutes of Health͞National Institute adult mammalian brain are derived from radial glia, which are on Aging. A.D.T. was supported by Damon Runyon Cancer Research ultimately derived from neuroepithelial cells. Fund Grant DRG-1601.

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