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

Radial glia give rise to adult neural stem cells in the subventricular zone Florian T. Merkle*†, Anthony D. Tramontin*†, Jose´ Manuel Garcıá-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

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