Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2061-2065, March 1995 Neurobiology Radial glial cell transformation to astrocytes is bidirectional: Regulation by a diffusible factor in embryonic forebrain KIM E. HUNTER AND MARY E. HATrEN Laboratory of Developmental Neurobiology, The Rockefeller University, 1230 York Avenue, New York, NY 10021 Communicated by Torsten N. Wiesel, The Rockefeller University, New York, NY, October 26, 1994 ABSTRACT During development of mammalian cerebral been thought to be a partially committed cell, with further cortex, two classes of glial cells are thought to underlie the differentiation toward an astrocyte cell fate occurring in a establishment of cell patterning. In the embryonic period, progressive manner. migration ofyoung neurons is supported by a system of radial To develop an in vitro model system for analysis of signals glial cells spanning the thickness of the cortical wall. In the that regulate the astroglial developmental pathway, we marked neonatal period, neuronal function is assisted by the physio- radial glial cells in situ so that they could subsequently be logical support of a second class of astroglial cell, the astro- identified in vitro, and we examined the control of process cyte. Here, we show that expression of embryonic radial glial formation and antigen marker expression by labeled cells. We identity requires extrinsic soluble signals present in embry- show that expression of a radial glial cell identity in mammalian onic forebrain. Moreover, astrocytes reexpress features of forebrain is determined by the availability of diffusible induc- radial glia in vitro in the presence of the embryonic cortical ing signals. These signals are temporally regulated during signals and in vivo after transplantation into embryonic development and are found in embryonic but not mature neocortex. These findings suggest that the transformation of forebrain. Furthermore, we show that these signals act to radial glia cells into astrocytes is regulated by availability of transform mature astrocytes to a radial glial phenotype, inducing signals rather than by changes in cell potential. indicating that the transformation from radial glial cell to astrocyte is reversible. Preliminary biochemical characteriza- In the developing brain, astroglial cells are among the first cells tion indicates that the inducing signals are protein in nature to differentiate, with bipolar radial glia being predominant in and may represent one or more previously uncharacterized the embryonic period. Described over a century ago by neural growth regulators. These findings provide support for Kolliker (1), Retzius (2), and Ramon y Cajal (3), radial glia the role of extrinsic signals in determining and maintaining a have been proposed to function in the initial steps of brain radial glial identity. histogenesis, including support of neuronal migration and laminar patterning (3-7). During the perinatal period, changes in astroglial form occur, with bipolar radial glia disappearing MATERIALS AND METHODS and being replaced by multipolar astrocytes. Differential Embryonic Cell Cultures. Embryonic day 14 (E14) mouse expression of cellular antigen markers by these two general forebrains were removed, placed in Earle's balanced salt classes of astroglia in the perinatal mammalian forebrain solution (EBSS; GIBCO), labeled pially with 1,1'-dioctadecyl- (8-10), and direct observation of successive changes in glial 3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI; Mo- form during later phases of histogenesis, has provided support lecular Probes; 3 mg/ml in EtOH/dimethyl sulfoxide), and for the general view that radial glial cells transform into then incubated in EBSS for 3 hr in 95% 02 at 32°C. Neocor- astrocytes (8-11). tices were dissociated by treatment with trypsin (17) and plated A general model for cell specification in the nervous system, on poly(L-lysine) (0.01 mg/ml; Sigma) at densities of 105-106 termed neuropoiesis, has emerged from studies of the neural cells per ml. For ceilings, 106 cortical cells from animals aged crest lineage, in which soluble factors control the expansion of E14 to P6 (postnatal day 6) were plated into membrane filter multipotent stem cell populations and restrict cell fate via a tissue culture inserts (Nunc). Inserts were overlaid onto cul- progressive restriction of gene expression (for reviews, see refs. tures of 105 DiI-labeled neocortical cells in DMEM-F12 12-14). Support for the existence of multipotent central nervous (GIBCO) supplemented with penicillin-streptomycin and system (CNS) neural precursor cells in vivo has been gained 0.4% ReduSer II (GIBCO) and maintained at 35°C in 5% through in vivo lineage tracing methods (15, 16). Within CNS C02/95% air. After 24 hr, cultures were fixed (2% paraformal- neural precursor cell populations, bipotential cells are thought to dehyde in phosphate-buffered saline), mounted in Slowfade generate neurons and glia, with the emergence of the radial glial (Molecular Probes), and then immunostained [mouse anti- lineage occurring early in cortical development (7). RC2 (gift of M. Yamamoto, University of Tsukuba, Japan) A critical question concerning the role of extrinsic signals in followed by fluorescein isothiocyanate (FITC)-conjugated neuropoiesis is whether differentiation factors are instructive goat anti-mouse IgM (Sigma)]. Cells were viewed by epifluo- or permissive. Instructive signals would commit a precursor rescence on a Zeiss Axiophot microscope. Coverslips were cell to a particular cell lineage at the expense of production of scored for the percentage of all Dil-positive cells bearing other classes of cells, while in a permissive system the gener- processes; 100 cells on each coverslip were scored in each case. ation of different classes of cells would follow a stochastic Preparation of Astrocyte Cultures and Assay of E14 Activ- mechanism, with differentiation factors permitting the sur- ity. Astrocytes were purified from P6 murine cerebral cortex vival of particular, partially committed progenitor cells (12). (17). Purified astrocytes were plated onto coverslips coated in Both cases imply a series of steps that progressively restrict the poly(L-lysine) within 24-well plates at a density of 104 cells per fate of precursor cells, with terminal differentiation of a ml. Astrocytes were cultured with or without an insert (pre- particular cell type. In classical studies, the radial glial cell has pared as described above) of 106 E14 neocortical cells for 48 The publication costs of this article were defrayed in part by page charge Abbreviations: CNS, central nervous system; E14, embryonic day 14; payment. This article must therefore be hereby marked "advertisement" in P6, postnatal day 6; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl- accordance with 18 U.S.C. §1734 solely to indicate this fact. indocarbocyanine perchlorate; GFAP, glial fibrillary acidic protein. 2061 Downloaded by guest on September 27, 2021 2062 Neurobiology: Hunter and Hatten Proc. NatL Acad ScL USA 92 (1995) hr and then fixed in 2% paraformaldehyde permeabilized in DMEM-F12 supplemented with penicillin-streptomycin. After ethanol, and immunostained for glial fibrillary acidic protein 30 min, collection of 50 2-ml samples began. Astrocytes were (GFAP) [rabbit anti-cow GFAP (Dako) followed by rhoda- cultured with 1 ml of each fraction for 2 days before immu- mine anti-rabbit IgG (Sigma), or RC2 followed by FITC- nostaining for GFAP and scoring for astrocyte morphology, as conjugated anti-mouse IgM (Sigma)] and then mounted in described above. Slowfade. Approximately 200 cells on each coverslip were In Utero Transplantations. Pregnant mice of 15 days ges- scored according to phenotype as normal or radial; the tation were anesthetized (375 jig of tribromoethyl alcohol per percentage of radial cells on each coverslip was calculated. g of body weight in 2.5% tertiary amyl alcohol) (Sigma). Western Blot Analysis of GFAP Levels. Astrocytes were Astrocytes harvested from P6 cortexwere labeled with PKH26 plated at subconfluent density into two T75 tissue culture as described (20) and resuspended at 20 x 106 cells per ml. flasks (Nunc) precoated with poly(L-lysine) in DMEM-F12 After laparotomy, the uterus was transilluminated to visualize with 10% fetal calf serum. On the following day, cells were embryos, and astrocytes (0.5 ,ul) were injected into the lateral washed twice in DMEM-F12 supplemented with ReduSer and ventricle of each embryonic forebrain with a 1-,ul Hamilton then aspirated; one flask was given 10 ml of serum-free DMEM-F12; the other was given 10 ml of E14 neocortical cell syringe, after which the injection site was sealed with cyano- conditioned medium. After 2 days, astrocytes were rinsed acrylic adhesive, the embryos were replaced in the abdominal twice in calcium and magnesium-free phosphate-buffered cavity, and the abdominal wall was sutured. Two days later, saline (GIBCO) and harvested with a cell scraper. Cells were embryos were perfused with 4% paraformaldehyde, and the pelleted and resuspended in 500 ,ul of protein sample buffer cortex was removed and postfixed for 24 hr. Cortices were (18), and then protein concentrations were determined (pro- embedded in 3% agarose; 100-,um coronal Vibratome sections tein detection kit; Bio-Rad). Samples were boiled and 5 or 20 were cut and mounted in Slowfade. Dye-labeled cells within ,ug was loaded onto a 12% acrylamide gel alongside Kaleido- Vibratome sections of transplant tissue were imaged with a scope molecular weight