308 nature neuroscience • volume 5 no 4 • april 2002 articles Cell diversity in the vertebrate nervous system is generated through a developmentally regulated cell fate restriction 1,2 . In the cerebral cortex, precursor cells are multipotent at early stages of development 3 and become specified to generate a single cell type at later stages 4,5 . These distinct lineages have been observed under isolated in vitro conditions 3,6–8 and show characteristic stereotyped lineage trees 8 , implying that cell-intrinsic determi- nants act as fate-restrictive cues. The transcription factor Pax6, which is expressed during neu- rogenesis in the cerebral cortex and is crucial for patterning the telencephalon 9–11 , is a possible intrinsic fate determinant. Pax6 is detected specifically in radial glial cells. In Small-eye (Sey) mice, which lack functional Pax6, these cells have a distorted mor- phology as well as altered gene expression patterns and cell cycle characteristics 10,12 . As radial glial cells of the cerebral cortex gen- erate neurons in vitro and in vivo 13–16 , we hypothesized that Pax6 might be involved in the neurogenic potential of radial glial cells. We used loss- and gain-of-function approaches to address this question. We show here that the neurogenic progeny of radial glial cells was reduced in the Pax6 mutant cortex, whereas Pax6 transduction enhanced the neuronal lineage and instructed even astrocytes towards neurogenesis. RESULTS Reduced neurogenic radial glia in Pax6 mutant To determine the progeny of radial glial cells in the Pax6 loss- of-function condition, we isolated radial glial cells by fluores- cence-activated cell sorting (FACS) using green fluorescent protein (GFP) expression driven by the human GFAP pro- Glial cells generate neurons: the role of the transcription factor Pax6 Nico Heins 1 , Paolo Malatesta 1 , Francesco Cecconi 2 , Masato Nakafuku 3 , Kerry Lee Tucker 4 , Michael A. Hack 1 , Prisca Chapouton 1 , Yves-Alain Barde 2 and Magdalena Götz 1 1 Max-Planck Institute of Neurobiology, Am Klopferspitz 18a, 82152, Planegg-Martinsreid, Munich, Germany 2 Dipartimento di Biologia, Universita degli studi ‘Tor Vergata’, Via della Ricerca Scientifica, 00133 Rome, Italy 3 University of Tokyo, Graduate School of Medicine, 7-3-1 Hongo, Bunkyoku, Tokyo, 113-0033 Japan 4 Friedrich Miescher Institute for Biomedical Research, Maulsbeerstr. 66, 4058 Basel, Switzerland P.M. and N.H. contributed equally to this work Correspondence should be addressed to M.G. (mgoetz@neuro.mpg.de) Published online: 18 March 2002, DOI: 10.1038/nn828 Radial glial cells, ubiquitous throughout the developing CNS, guide radially migrating neurons and are the precursors of astrocytes. Recent evidence indicates that radial glial cells also generate neurons in the developing cerebral cortex. Here we investigated the role of the transcription factor Pax6 expressed in cortical radial glia. We showed that radial glial cells isolated from the cortex of Pax6 mutant mice have a reduced neurogenic potential, whereas the neurogenic potential of non- radial glial precursors is not affected. Consistent with defects in only one neurogenic lineage, the number of neurons in the Pax6 mutant cortex in vivo is reduced by half. Conversely, retrovirally mediated Pax6 expression instructs neurogenesis even in astrocytes from postnatal cortex in vitro. These results demonstrated an important role of Pax6 as intrinsic fate determinant of the neurogenic potential of glial cells. moter 13,17 . The hGFAP–GFP transgene was crossed into the Pax6 mutant background using Sey mice that express a trun- cated, non-functional form of Pax6 (ref. 18). GFP-positive cells from wild-type and homozygous Pax6 mutant (Sey/Sey) lit- termates were analyzed by FACS (Fig. 1a–d). Consistent with previous sorting results, most GFP-positive cells sorted from the cortex of embryonic day (E)14 wild-type and Sey/Sey mice were precursors immunoreactive for nestin and the radial glial antigens RC2 and GLAST (Fig. 1c,d, gray). In wild-type cortex, sorted radial glial cells were also Pax6 immunoreactive (78 ± 4% of sorted RC2-positive cells, n = 159, data not shown). Besides radial glial cells, a relatively high proportion of postmitotic neu- rons labeled with neuron-specific antiserum against β-tubulin- III was included in the GFP-positive fraction (Fig. 1c,d; 2 hours). This most likely was due to the remaining GFP signal in the young neuronal descendants of the sorted radial glia 13 . How- ever, GFP-positive cells were reduced in the Pax6 mutant mice compared to their wild-type littermates (Fig. 1a,b; p < 0.005, 4 litters). Thus, GFP is targeted to the same populatiozn of GLAST-positive radial glia in Pax6 mutant mice, but their pro- portion is lower than in wild-type mice. The progeny of the GFP-positive cells sorted from wild-type and Sey/Sey cortex was examined after 1 week in vitro by double staining with cell- type specific antisera as described previously 13 (see also Fig. 1 legend). Pax6 mutant radial glia generated a significantly small- er number of pure neuronal clones within 1 week in vitro as compared to wild-type littermates (Fig. 1c,d; blue fraction; for example, Fig. 1e, g). Correspondingly, more non-neuronal clones were generated by the GFP-positive precursors from © 2002 Nature Publishing Group http://neurosci.nature.com