During development, radial glial cells displaying a bipolar shape span their processes along the full width of the cerebral wall. Radial glial somata are mainly situated in the ventricular or subventricular zones. Their short processes are anchored in the ventricular zone and their elongated processes reach the pial surface. The long radial processes are known to serve as a scaffold for neuroblasts to migrate from the (sub)ventricular zone to their final destination, such as the cortical plate. After neuronal migration has ceased, transformation of radial glial cells into astrocytes occurs. Radial glial cells may reenter mitosis and differentiate into different types of astrocytes.

The process of transformation of radial glial cells into astrocytes, which takes place over a considerably prolonged period of time in the human developing brain, encompasses detachment from the ventricular surface, outward displacement of the cell body and loss of radial orientation. Further morphological changes are found at the elongated process of radial glial cells during transformation. The processes appear fragmented and display varicosities. Regularly, processes have been observed to terminate at blood vessels.

A number of experimental studies have addressed questions concerning the regulation of the phenotype, and maintenance and transformation of radial glial cells. In these studies, various regulatory mechanisms have been identified which involve a wide range of signaling molecules, for example Notch, the transcription factor PAX6, transforming growth factor (TGF-), brain lipid binding protein (BLBP), ciliary neurotrophic factor (CNF), leukemia inhibitory factor (LIF), epidermal growth factor (EGF) receptor and reelin, the latter of which is secreted by Cajal-Retzius cells. Some of these factors may also be involved in the generation of neurons from radial glial cells. This role of radial glial cells as neuronal precursors has recently been demonstrated in a number of studies.

Sphingosine-1-phosphate (S1P), which is a sphingolipid mainly derived from platelets, has been shown to induce proliferation of astrocytes. The main route of S1P synthesis involves the phosphorylation of sphingosine by sphingosine kinase. The biological activities of S1P, which include cytoskeletal reorganization and regulation of cell motility and cell growth, are now considered to be mediated by the endothelial differentiation gene (Edg)-family of G protein-coupled receptors. At present eight Edg receptors (Edg-1-8) have been identified of which Edg-1, Edg-3, Edg-5, Edg-6 and Edg-8 are specific receptors for S1P and Edg-2, Edg-4 and Edg-7 are activated by lysophosphatidic acid. Northern blot analysis showed Edg-8 transcripts to be almost exclusively present in the central nervous system with an ubiquitous expression in different brain regions and the spinal cord.

The objective of the present study was to investigate expression patterns of Edg-8 in the human fetal brain with special reference to radial glial cells. To elucidate a putative association of Edg-8 to radial glia, double labelling was performed using the intermediate filament protein vimentin as a marker for radial glial cells.

Twelve human fetal brains without neuropathological alterations were used in the present study. The age of the fetuses, which were obtained from legal elective or spontaneous abortions, ranged from 12 to 38 weeks of gestation (wg). After detachment of the brain stem from the whole brain, the cerebral hemispheres were separated by a medio-sagittal cut. One hemisphere of each brain was cut into frontal blocks, which were fixed in 3.7% paraformaldehyde and cryoprotected in 0.05 M Tris-buffered saline (TBS, pH 7.4) containing 30% sucrose. From these blocks, 120 ?m-thick sections were cut using a cryostat. The sections were rinsed in TBS and handled in a free-floating manner.

To reduce non-specific immunostaining sections were pre-treated with: (1) 10% methanol and 7% hydrogen peroxide (H₂O₂) followed by (2) 1.5% lysine, 0.25% Triton X-100 and 10% bovine serum albumin. Then, sections were incubated with anti-Edg-8 (rabbit, anti-human; Zytomed, Berlin, Germany; diluted 1:250) at 4 °C for approximately 40 h. Thereafter, biotinylated anti-rabbit IgG (diluted 1:200; Vector Laboratories, Burlingame, CA, USA) was applied for 2 h. Sections were then incubated with the avidin–biotin–peroxidase complex for another 2 h followed by visualization of the immunocomplex using 0.07% diaminobenzidine (DAB) and 0.003% H₂O₂.

For double-labelling, sections were first stained for Edg-8 using DAB according to the procedure described above. These sections were then incubated with an anti-vimentin monoclonal antibody (mouse; Sigma-Aldrich, Deisenhofen, Germany; diluted 1:40) or an anti-O4 anti-human antibody that is specific for oligodendrocytes (mouse; Chemicon, Temecula, CA, USA; diluted 1:100) at 4 °C for approximately 40 h. Afterwards, alkaline phosphatase-conjugated anti-mouse secondary antibody (diluted 1:500; Vector Laboratories) was applied for 2 h. Alkaline phosphatase activity was visualized with the blue alkaline phosphatase substrate kit III (Vector Laboratories).

For controls, the primary antibody was omitted in every tenth section.

Until 22 wg, radial glial fibers did not display Edg-8 immunostaining. At 20 and 22 wg Edg-8-positive cells are encountered in the globus pallidus, the putamen and the adjacent medullary laminae. Double labelling revealed that these multipolar Edg-8-immunostained cells were also O4 positive (data not shown); thus, these cells most likely are oligodendrocytes or their precursors.

At 24 wg, Edg-8-immunostained structures were observed in allocortical areas (i.e. entorhinal cortex and hippocampal formation). In the cortical plate and upper subplate of these areas, discontinuously labelled fibers were present that were aligned radially. At higher magnification, a large number of varicosities were detected, in particular in the marginal zone and cortical plate. After Edg-8/vimentin double-labelling, it was evident that radial glial fibers expressed Edg-8 as well as vimentin; at high magnification, it appeared that segments of radial glial fibers were vimentin-positive and in between these segments short Edg-8-positive fragments were present. Only occasionally, short fragments were double labelled for Edg-8 and vimentin. The radial glial fibers, which revealed a moderate packing density, often appeared undulated. Vimentin- as well as Edg-8-immunolabelled fibers were found to terminate on vessels. Edg-8 staining was not be detected in soma, the short processes or the proximal part of elongated process. These parts of the radial glial cells were only vimentin-positive.

In a recent study, Edg-8 receptors have been demonstrated to be preferentially expressed in the oligodendrocyte cell lineage in the rat central nervous system. Thus, Edg-8 receptors (and their ligand S1P) have been implicated to be involved in oligodendrocyte maturation and myelin formation. In line with this finding, a considerable number of Edg-8-positive cells have been found in the human developing forebrain in the present study. The distribution pattern of these cells closely parallels the progress of myelin formation in the human fetal forebrain as reported in the literatureUlfig et al., 1998 N. Ulfig, J. Nickel and U. Saretzki, Alterations in myelin formation in fetal brains of twins, Pediatr Neurol 19 (1998), pp. 287–293. Article | PDF (743 K) | View Record in Scopus | Cited By in Scopus (8). Moreover, these Edg-8-positive cells coexpress the oligodendrocyte marker O4. Thus, they are most probably immature or mature oligodendrocytes.

As far as we know, the results presented here demonstrate for the first time that the G protein-coupled receptor Edg-8 is expressed in radial glial fibers. This finding is based on a number of observations: firstly, Edg-8 was detected in discontinuous radially aligned fibers with numerous varicosities which closely correspond to the appearance of vimentin-positive radial glial fibers in the upper cerebral wall as described by Ulfig et al. (1999). Additionally, Edg-8-positive fragments were associated with vimentin-positive radial glial fibers after double-labelling. From the eighth gestational month onwards, Edg-8-positive radial glial fibers disappear and are gradually replaced by Edg-8-positive mono- and bipolar glial cells. The latter most likely represent transitional stages between radial glial cells and astrocytes characterized by detachment of radial processes and an outward displacement of cell somata.