The tripotential glial-restricted precursor (GRP) cell and glial development in the spinal cord: generation of bipotential oligodendrocyte-type-2 astrocyte progenitor cells and dorsal-ventral differences in GRP cell function

Abstract

We have found that the tripotential glial-restricted precursor (GRP) cell of the embryonic rat spinal cord can give rise in vitro to bipotential cells that express defining characteristics of oligodendrocyte-type-2 astrocyte progenitor cells (O2A/OPCs). Generation of O2A/OPCs is regulated by environmental signals and is promoted by platelet-derived growth factor (PDGF), thyroid hormone (TH) and astrocyte-conditioned medium. In contrast to multiple observations indicating that oligodendrocyte precursor cells in the embryonic day 14 (E14) spinal cord are ventrally restricted, GRP cells are already present in both the dorsal and ventral spinal cord at E13.5. Ventral-derived GRP cells, however, were more likely to generate O2A/OPCs and/or oligodendrocytes than were their dorsal counterparts when exposed to TH, PDGF, or even bone morphogenetic protein-4. The simplest explanation of our results is that oligodendrocyte generation occurs as a result of generation of GRP cells from totipotent neuroepithelial stem cells, of O2A/OPCs from GRP cells and, finally, of oligodendrocytes from O2A/OPCs. In this respect, the responsiveness of GRP cells to modulators of this process may represent a central control point in the initiation of this critical developmental sequence. Our findings provide an integration between the earliest known glial precursors and the well-studied O2A/OPCs while opening up new questions concerning the intricate spatial and temporal regulation of precursor cell differentiation in the CNS.

Fig. 1.

GRP-derived O4⁺ cells are bipotential and represent O2A/OPC-like cells. Freshly isolated GRP cells were grown for 3 weeks in defined medium in the presence of bFGF and then switched to a medium supplemented with bFGF and TH; after 5 d, cultures were stained with the O4 antibody (see Materials and Methods). Cells were then dislodged from the surface and plated at clonal density in poly-l-lysine-coated dishes. Single O4⁺ cells were circled. After 3 d in culture, cells were exposed to medium supplemented with bFGF and 10% FCS. (Parallel experiments using BMP4 instead of FCS yielded identical results.) After 5 d, clones were stained with A2B5 (rhodamine), anti-GFAP (fluorescein), and anti-GalC (coumarin) antibodies. The coumarin staining is not shown because none of the clones contained any GalC⁺ oligodendrocytes in this condition.A, Clone derived from a single O4⁺GalC⁻ cell. The progeny from O4⁺ founder cells consists exclusively of A2B5/GFAP double-positive cells, consistent with the antigenic phenotype of type-2 astrocytes. B, Clone derived from a single O4⁻GalC⁻ cell. The progeny from O4⁻ founder cells consists of A2B5/GFAP double-positive type-2 astrocytes and A2B5⁻GFAP⁺ cells, representing type-1 astrocytes (indicated by arrows).

Fig. 2.

Both dorsal- and ventral-derived GRP cells generate O4⁺GalC⁻ cells. A2B5⁺ cells were isolated from either the dorsal or ventral spinal cord of E13.5 rat embryos and plated in the presence of bFGF supplemented with PDGF plus TH (P + TH) for 7 d. Dorsal and ventral cultures were then stained with O4, anti-GalC, and anti-GFAP antibodies. Both dorsal- and ventral-derived cultures generated comparable numbers of O4⁺precursor cells. However, GalC⁺ oligodendrocytes were found predominantly in ventral-derived GRP cell cultures. Only a small fraction of both dorsal and ventral cultures gave rise to GFAP⁺ astrocytes. Two independent experiments examining six data points for each condition revealed comparable results.

Fig. 3.

BMP4 induces differentiation of astrocytes from dorsal- and ventral-derived A2B5⁺ cells in a dose-dependent manner. A2B5⁺ cells, isolated from either the dorsal or ventral spinal cord of E13.5 rat embryos, were plated at a low density in the presence of bFGF and increasing concentrations of BMP4 (0.1–100 ng/ml). After 3 d, cultures were labeled with anti-GFAP antibodies and the number of astrocytes was determined. Whereas dorsal cultures exhibited a continuous, dose-dependent increase in the number of GFAP⁺astrocytes, ventral-derived GRP cells generated significantly more astrocytes at lower doses of BMP (1 ng/ml) at this time point, and higher doses of BMP4 (100 ng/ml) proved to be lethal to ventral-derived GRP cells.

Fig. 4.

Differential effects of BMP4 on dorsal- and ventral-derived GRP cells. GRP cells were isolated from either the dorsal or ventral spinal cord of E13.5 rat embryos and plated at a low density in the presence of FGF and BMP4 (A) (1 ng/ml) or FGF, BMP4 (1 ng/ml), and TH (B). To allow for oligodendrocyte generation, cultures were examined after 7 d for the presence of O4⁺GalC⁻ precursor cells or GalC⁺ oligodendrocytes. Although GalC⁺ oligodendrocytes were only found in ventral GRP cell cultures containing TH, both dorsal- and ventral-derived cultures contained O4⁺GalC⁻precursor cells. In the presence of BMP, the ability of dorsal GRP cells to generate O4⁺GalC⁻precursor cells was lower than that of ventral-derived cultures; this was not changed by the addition of TH.

Fig. 5.

Sequential lineage restriction in the glial lineage of the CNS. A side-by-side comparison of the salient features of two lineage-restricted glial precursors of the CNS is shown. The evidence presented here strongly suggests a progressive and sequential transition from the tripotential GRP cell to the bipotential O2A/OPC. In the developing spinal cord, it currently seems most likely that this transition is controlled in a temporal and spatial pattern and is regulated by cell-extrinsic signaling molecules (Pringle et al., 1992; Rao and Mayer-Pröschel, 1997; Rao et al., 1998).