Proteolipid promoter activity distinguishes two populations of NG2-positive cells throughout neonatal cortical development

Abstract

Transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the mouse myelin proteolipid protein (PLP) gene promoter have been developed to investigate cells in the oligodendrocyte lineage. Transgene expression is consistent with the developmental expression of PLP, with cells at all stages of oligodendrocyte differentiation clearly visualized. These animals were analyzed to establish the time course of oligodendrocyte progenitor migration, proliferation, and differentiation in neonatal cortex. In these animals, two populations of NG2 proteoglycan-positive oligodendrocyte progenitor cells were identified that exist in postnatal subventricular zone and cortex. These two populations are distinguished by the presence or absence of PLP gene expression. Thus, PLP gene expression defines a subpopulation of NG2-positive cells from very early postnatal ages, which migrates toward the pial surface and proliferates in situ. EGFP(+)/NG2(+) cells are present in the cortex from postnatal day 1, and they remain in the cortex as undifferentiated oligodendrocyte progenitors for up to 3 weeks before myelination begins. These data could be explained by the presence of an important inhibitor of oligodendrocyte differentiation in the cortex during this period, which is downregulated in a region-specific manner to allow myelination. On the other hand, it is possible that oligodendrocyte progenitor cells remain undifferentiated in cortex until an essential signal is produced in situ to induce differentiation.

Fig. 1.

Overview of EGFP expression in postnatal nervous system. A, Coronal section of P21 brain (EGFP10) showing vivid fluorescence in white matter tracts, including corpus callosum (cc), caudate putamen (cp), anterior commissure (ac), and the lateral olfactory tract (lot). B, Sagittal section of P21 brain (EGFP5) demonstrating strongest fluorescence in the brainstem and spinal cord, as well as white matter tracts of the cerebellum (Cb). C, Cross section of P30 spinal cord (EGFP3) showing EGFP- positive cell bodies in both gray and white matter.D, Teased fibers of 6 month sciatic nerve (EGFP10) showing fluorescence in cell bodies (arrowhead) and the paranode (arrow). Scale bars: A, 1000 μm; B, 2000 μm; C, 500 μm;D, 25 μm.

Fig. 2.

Transgene expression is found in PNS and CNS of embryos from E10.5. A, Sagittal cryostat section of E10.5 embryo (EGFP5) showing fluorescence in the basal plate of the diencephalon in the developing brain and the dorsal root ganglia of the PNS (arrow). B, Sagittal cryostat section of E14.5 embryo (EGFP5) showing fluorescence around the olfactory bulb (Olf), in the diencephalon (Di), and the spinal cord. Transgene expression is also apparent in PNS structures, including the digits (Digits). C, Axial cryostat section of E14.5 embryo spinal cord (EGFP5) demonstrating the mainly ventral localization of transgene expression. Some EGFP-positive cells are visible in the dorsal region. DRG, Dorsal root ganglion. Scale bars: A, B, 1000 μm; C, 250 μm.

Fig. 3.

Transgene expression may be detected at all stages of the oligodendrocyte lineage. A, EGFP-stained myelinating oligodendrocyte in P21 brain (EGFP5). Note the many parallel processes of myelinated axons. B, NG2 immunostaining of P22 cortex (EGFP10) showing oligodendrocyte progenitor cells. Some NG2-positive cells (Texas Red) are clearly expressing the transgene and are often found in closely apposed pairs or doublets. C, PLP/DM20 (Texas Red) immunostaining of P1 subcortical white matter (EGFP10) showing a clear band of premyelinating oligodendrocytes only in the developing subcortical white matter. D, PLP/DM20 (Texas Red) immunostaining of P1 corpus callosum (EGFP10). Cells in an apparent progression of differentiated states are numbered 1–5, as discussed in Results. Scale bars: A, B,D, 25 μm; C, 50 μm.

Fig. 4.

EGFP and plp/DM20 expression in developing cortex. A, Fluorescence imaging of EGFP expression from P1 to 8-week-old animals (EGFP10) in medial frontal sections of cortex (midline is on the left). Intensity of fluorescence increased as the cells became obviously myelinating cells, although cell density did not increase. Arrows in the 8 week sample identify EGFP-positive cells that are also NG2-positive (data not shown). Scale bars, 100 μm. B, PCR analysis of plp (top band) and DM20(bottom band) transcripts in cDNA (+) or reverse transcriptase-negative RNA (−) from cortex dissected from P1–P10 animals (EGFP5). DM20 transcripts are the most abundant in the early ages, including E16.5 whole brain.

Fig. 5.

Radial migration through the gray matter is complete by P6. A, Many EGFP-positive cells in the outer cortex of P1 brain appear to have a migratory morphology, with some exhibiting a unipolar morphology (EGFP10). B, NG2 immunostaining of P1 outer cortex (EGFP10) showing that the EGFP-positive cells in P1 cortex also express NG2 (Texas Red).Arrowheads indicate nongreen NG2-positive cells.C, By P4, the cells in the outer cortex are beginning to show a few doublets, suggestive of proliferation (seearrows), but some still retain the unipolar shape (EGFP10). D, At P6, the cells populating the outer cortex have lost the unipolar leading process and are often found in pairs (arrowheads; EGFP10). All sections are oriented comparably, with the pial surface at the top right in the image (PIA). Scale bars: A,C, D, 50 μm; B, 25 μm.

Fig. 6.

EGFP⁺/NG2⁺cells are capable of division. A, An oligodendrocyte progenitor cell, positive for the transgene, has retracted processes and upregulated NG2 immunoreactivity (Texas Red, EGFP10). This cell also exhibits a small intense region of BrdU staining (Cy5; seearrow in the spliced image) after 4 hr BrdU labeling, suggesting that this cell may be about to undergo mitosis. Separate channel images are shown in the corners.B, A doublet of EGFP⁺/NG2⁺ cells that also stain for BrdU after 8 hr labeling, indicating that these cells have recently undergone, or are currently undergoing, mitosis (EGFP10).C, EGFP⁺/NG2⁺/BrdU⁺cells expressed as a percentage of total EGFP⁺/NG2⁺ cells shows that the peak of division of this cell population is at approximately P4.D, Both populations of NG2⁺/BrdU⁺ cells are expressed as a percentage of total BrdU⁺ cells, showing that NG2⁺ cells become the predominant proliferating cell type after P1 and that the ratio of proliferating EGFP-positive to EGFP-negative/NG2⁺ cells remains relatively constant with age. Scale bars: A, 10 μm; B, 5 μm.

Fig. 7.

EGFP⁺/NG2⁺cells are present in the subventricular zone at P1. P1 tissue (EGFP10) was stained for NG2 proteoglycan and imaged for NG2 (Texas Red) and EGFP. A, Image of general SVZ area. LV , Lateral ventricle. B, Higher magnification of cells near the SVZ. Arrows highlight EGFP⁻/NG2⁺ cells in the SVZ. Scale bars, 10 μm.