Type-2 Cells as Link Between Glial and Neuronal Lineage in Adult Hippocampal Neurogenesis BARBARA STEINER, 1,2  FRIEDERIKE KLEMPIN, 1,2  LIPING WANG, 1 MONIKA KOTT, 1 HELMUT KETTENMANN, 1 AND GERD KEMPERMANN 1,2 * 1 Max-Delbr € uck-Center for Molecular Medicine (MDC) Berlin-Buch, 13125 Berlin-Buch, Germany 2 Volkswagenstiftung Research Group, Department of Experimental Neurology, Charit e University Medicine Berlin, 10117 Berlin, Germany KEY WORDS precursor cells; BLBP; radial glia; dentate gyrus; Sox2 ABSTRACT In the course of adult hippocampal neurogenesis, new cells go through a series of stages associated with proliferative activity. The most highly proliferative cell type is an inter- mediate precursor cell, called type-2 cell. We here report that on the level of type-2 cells a transition takes place between features associated with the glial and the neuronal lineage. We show that stem-cell marker Sox2 and radial glia marker BLBP are expressed in type-2 cells but label only a small percentage of the proliferating cells. By and large, precursor cell marker Sox2 was found to be widely expressed in hippocampal astrocytes. Between 3 h and 1 week after a single injection of permanent S-phase marker bromodeoxyuridine (BrdU), the number of BrdU-labeled BLBP-positive cells did not change, consistent with the idea that both markers here are associated with the maintained precursor cell pool. Using reporter gene mice expressing the green fluorescent protein (GFP) under the promoter for nes- tin we found an overlap of GFP with markers of the neuronal lineage, doublecortin (DCX) and transcription factor NeuroD1 in type-2 cells, whereas in glial fibrillary acidic protein (GFAP)-GFP mice expression of GFP and NeuroD1 or DCX was mutually exclusive. Electrophysiologically, the group of type-2 cells fell into two subgroups: one with astrocytic prop- erties and another with an early ‘‘complex’’ phenotype of neural progenitor cells. Our data further support the exis- tence of proliferative precursor cells that mark the transition between glia-like states and neuronal differentiation. V V C 2006 Wiley-Liss, Inc. INTRODUCTION In adult hippocampal neurogenesis, new granule cells are generated from precursor cells residing in the sub- granular zone (SGZ) of the dentate gyrus (Alvarez- Buylla and Lim, 2004; Kempermann et al., 2004). Simi- lar to other stem-cell systems, such as bone marrow, skin, or intestines, a hierarchy of cells with stem-cell properties exist in the SGZ. Seri and Alvarez-Buylla identified a radial glia-like astrocyte as the stem cell of the adult hippocampus (Seri et al., 2001, 2004). We later described how adult neurogenesis proceeds from this cell over intermediate progenitor stages to postmitotic matu- ration (Brandt et al., 2003; Filippov et al., 2003; Kem- permann et al., 2004; Steiner et al., 2004). Although abundant, the radial glia-like stem cell (type-1) is hardly dividing (Filippov et al., 2003) but its proliferation can be stimulated, e.g., by hypoxia or seizures (Huttmann et al., 2003; Kunze et al., 2006). Most of the proliferative activity in the adult SGZ is accounted for by type-2 cells as the transiently amplifying progenitor cells. Type-1 cells might constitute the largely quiescent population of stem cells that can replenish the pool of transiently amplifying progenitor cells (Seri et al., 2001, 2004). The concept of radial glia-like cells serving as stem cells in the adult hippocampus is in line with similar reports from fetal brain development (Heins et al., 2002; Malatesta et al., 2003; Noctor et al., 2001, 2002) and the adult subventricular zone (Doetsch et al., 1997; Scheffler et al., 2005). Type-2 cells were initially identified in nestin-GFP re- porter gene mice, in which expression of the green fluo- rescent protein (GFP) is driven by the neural enhancer element of the nestin promoter (Yamaguchi et al., 2000). Nestin is an intermediate filament expressed in many, if not all, neural precursor cells (Lendahl et al., 1990). Morphologically, type-2 cells are irregularly shaped cells with dense chromatin and brief, more or less horizontal processes. Electrophysiologically, the population of type- 2 cells appeared diverse and included cells with astrocy- tic properties like the type-1 cells, cells with the complex phenotype initially described for oligodendrocyte precur- sor cells, and a few cells with sodium currents and thus first neuronal properties (Filippov et al., 2003; Fukuda et al., 2003; Wang et al., 2005). A subpopulation of type- 2 cells was also shown to receive the first synaptic input, which is GABAergic (Wang et al., 2005). From these data it became apparent that type-2 cells mark a transition between cells with a glial phenotype (as far as the neural precursor cells are glial) and cells with features of the neuronal lineage. What remained unclear was the time-point of this transition. Grant sponsor: Volkswagenstiftung. *Correspondence to: Gerd Kempermann, M.D., Max-Delbr€ uck-Centre for Molecu- lar Medicine (MDC), Berlin-Buch, Robert-R€ ossle-Str. 10, 13125 Berlin, Germany. E-mail: gerd.kempermann@mdc-berlin.de B.S. and F.K. contributed equally to this work. Received 19 June 2006; Revised 13 July 2006; Accepted 24 July 2006 DOI 10.1002/glia.20407 Published online 6 September 2006 in Wiley InterScience (www.interscience. wiley.com). GLIA 54:805–814 (2006) V V C 2006 Wiley-Liss, Inc.