Elsevier

Neuroscience Research

Volume 41, Issue 1, September 2001, Pages 51-60
Neuroscience Research

Radial glia is a progenitor of neocortical neurons in the developing cerebral cortex

https://doi.org/10.1016/S0168-0102(01)00259-0Get rights and content

Abstract

Neocortical neurons are produced by cell division of neural stem cells in the ventricular zone of the cerebral cortex. We investigated the production of neurons by infecting neuroepithelial cells with a modified GFP-recombinant adenovirus. The adenovirus DNA is inherited by only one daughter cell at each cell division and travels one way from the progenitor to the progeny. Since the ventricular zone (VZ) of the embryo neocortex expressed an adenovirus receptor, CAR ubiquitously, morphology and cell-lineage of cells in the VZ could be revealed by the adenovirus infection. Radial glias, cells with a bipolar shape, and spherical cells were found as modified-GFP-positive (mGFP+) in the VZ. The bipolar cells (radial cells) had a radial process not in contact with the pia mater and a growth-cone-like structure at the edge of their radial process, while the radial glias had a process spanning all the cortical layers. Ten hours after viral infection, most mGFP+ cells were radial cells. In the following 8 h, the percentage of mGFP+ radial glias in mGFP+ neocortical cells increased from 18 to 50%, while that in radial/spherical cells decreased from 75 to 19%. The radial glias often divided asymmetrically and produced spherical cells and neuronal precursors. The spherical cells seemed to become radial cells by extending a radial process. The spherical cells, radial cells and radial glias seemed to constitute a proliferating cell cycle during which postmitotic neuronal precursors are produced. The neuronal precursors that inherited the radial processes migrated radially and developed into neocortical neurons. Four days after the viral infection, 97% of mGFP+ cells were neocortical neurons. Here, we propose that the radial glia is a progenitor of neocortical neurons, and that a significant number of radially migrating neurons is guided by their own radial processes connected to the pia mater.

Introduction

In the developing cerebral cortex, radial glias have been considered to play an important role in guiding radially migrating neurons from the ventricular zone (VZ) to the cortical plate and contribute to the specification of cerebral cortical areas (Rakic, 1972, Rakic, 1988, Rakic, 1990). Recently, however, radial glias were suggested to have proliferative activity to be considered as multipotent progenitor cells (Chanas-Sacre et al., 2000, Malatesta et al., 2000, Hartfuss et al., 2001, Noctor et al., 2001). The radial glial system is not static. Proliferation, extension of new fibers, and translocation of radial glial cell bodies are common features found in the radial glial system across the entire developing brain (Misson et al., 1988b, Misson et al., 1991, Voigt, 1989, Takahashi et al., 1990). Thus, it should be a very important issue to understand the in vivo behavior of radial glias in developing brains. In order to address this issue, first we should have a tool for identifying the radial glias in vivo. Although radial glias are immunoreactive for several molecular markers, such as RC2 (Misson et al., 1988a, Gaiano et al., 2000), vimentin (Kamei et al., 1998), and nestin (Lendahl et al., 1990, Gaiano et al., 2000, Super et al., 2000), these molecular markers are also used for identification of neural stem cells in vitro (Malatesta et al., 2000, Hartfuss et al., 2001) and in vivo (Yamaguchi et al., 2000). Furthermore, RC2 immunoreactivity was observed not only in radial glias but also in radial cells (Alvarez-Buylla et al., 1990), which are found in the VZ and had radial processes not spanning all the cortical layers (Gadisseux et al., 1992). Therefore, at present, no reliable molecular markers are available for the identification of the radial glia. Hence, we went back to the original definition of the radial glia, i.e. glial cells with a soma in the VZ and with a radial fiber spanning all the cortical layers (Rakic, 1972, Rakic, 1988, Rakic, 1990). In this study, we applied a recombinant adenovirus-expressing-membrane-targeted green fluorescent protein (mGFP) (Moriyoshi et al., 1996) to the lateral ventricle of the mouse embryo. This technique was revealed to be useful in investigating the detailed structures of the infected cells in the brain (Tamamaki et al., 2000). We infected neuroepithelial cells in vivo at the stage when most of the cortical neurons were produced and followed the fate of mGFP-positive (mGFP+) cells at various developmental stages. Here, we report that radial glias proliferate not only to produce glial progeny (Kamei et al., 1998, Misson et al., 1988b, Misson et al., 1991, Voigt, 1989), but also to supply neocortical neurons in vivo (Tamamaki, 2000).

Section snippets

Viral construction and infection

The present experiments were approved by the Committee for Animal Care and Use, and the committee for recombinant-DNA experiments of the Graduate School of Medicine in Kyoto University. AxCAG-GAP43-EGFP was constructed by inserting cDNA of GAP43 palmitoylation site and that of EGFP into the SwaI site in an adenovirus vector, pAxcw (Adenovirus kit, TAKARA, Japan). Then the recombinant adenovirus vector was introduced into 293 cells according to the manufacturer's protocol. After three

Results

To investigate the proliferation of neural stem cells and the production of postmitotic neurons, we injected recombinant adenovirus AxCAG-GAP43-EGFP into the lateral ventricle of embryos at E14–15, the stage when the layers V and IV neocortical neurons are produced (Fig. 1A). mGFP has a palmitoylation site at the N terminus, and is distributed on the plasma membrane to produce Golgi-stain-like labeling (Tamamaki et al., 2000). After the viral injection, the embryos were allowed to develop from

Discussion

Development of mGFP+ radial cells into radial glias and production of spherical cells by cell division of radial glias suggested that spherical cells, radial cells, and radial glias together constituted a proliferating cell cycle in the VZ (Fig. 4). Furthermore, the present data strongly suggested that radial glias are progenitors of neocortical neurons. Radial glias have long been considered to be nonproliferating supporting cells that serve as a guide for migrating neurons (Rakic, 1972,

Acknowledgements

We gratefully acknowledge the kind gift of Dr R. Kuwano. We also thank Uesugi for assistance in photography. This study was supported by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (Nos. 11170229, 11878151, 12053239, 12210085, 12053245, 12308039, and 12680731), and CREST of Japan Science and Technology.

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