NG2-expressing cells are only a proportion of Olig2-expressing cells
OPCs are a population of glial cells in the CNS which comprise about 5% of total cells and are the main proliferating cell type in the resting state of adult CNS . In the adult CNS, OPCs can differentiate into myelinating oligodendrocytes and are usually characterized as NG2 proteoglycan expressing cells. Therefore, we performed co-immunostaining of Olig2 and NG2 to study their relative distributions in the adult mouse CNS. Figure 1 illustrates the overlay of both Olig2 and NG2 signals across different brain regions (Fig. 1a, sagittal brain section at low magnification) and the spinal cord (SC) (Fig. 1n, coronal section) in an adult mouse. Clearly, while Olig2 and NG2 signal can be detected throughout the CNS regions, their relative expression level is not evenly distributed among different regions. One striking feature is the obvious strong signal of Olig2 (green) in the thalamus (TH) and midbrain (MB) (Fig. 1a). To more clearly understand the relationship between the NG2 and Olig2 expression pattern, we divided the brain into 12 discrete regions including the olfactory bulb (OB), anterior olfactory nucleus (AON), motor cortex (MO), striatum (STR), somatosensory cortex (SS), visual cortex (VIS), retrosplenial area (RSP), hippocampus (Hip), thalamus (TH), midbrain (MB), cerebellum (CB), and medulla (MY) (Fig. 1a). As shown in the high magnification confocal images of the brain (Fig. 1b-m) and SC (Fig. 1o), almost all the NG2-expressing cells in different CNS regions were clearly co-stained with Olig2, suggesting that all NG2 cells express Olig2. However, there were a considerable number of Olig2+ cells that did not have NG2, indicating a subpopulation of Olig2-expressing cells that are NG2 negative in the adult CNS. For example, in the thalamus and midbrain, only 13-16% of Olig2+ cells were NG2+ cells (see Table 1 for quantification). What are the majority of the Olig2-positive but NG2-negative cells in the thalamus and midbrain? What about the other CNS regions in the adult mice? To answer these questions, we further conducted a series of experiments to characterize the Olig2+ cells throughout various CNS regions.
Oligodendrocytes are only part of the Olig2-expressing cells
Oligodendrocytes are myelinating cells that are differentiated from OPCs under the control of bHLH transcriptional factor Olig2 . Besides OPCs, we also analyzed the expression pattern of Olig2 in oligodendrocytes by co-staining Olig2 with two typical markers of oligodendrocytes, CNPase and CC1 . CNPase, comprising about 4% of the total myelin protein in the CNS, is widely expressed in pre-myelinating and myelinating oligodendrocytes . We detected CNPase expression in the whole brain (Fig. 2a) and spinal cord (Fig. 2n), reflecting a uniform distribution of oligodendrocytes in the adult CNS. At high magnification, we found that all the CNPase-expressing oligodendrocytes had Olig2 immunofluorescence signal ( Fig. 2b-m, o). However, not every Olig2+ cells showed CNPase signal (Fig. 2b-m, o, Table 1), indicating the diversification of the proportion of oligodendrocytes in the adult CNS.
Next, we performed co-immunostaining of Olig2 with mature oligodendrocyte marker CC1. Unlike myelin protein CNPase, CC1 was highly localized in the soma of the oligodendrocytes (Fig. 3). Our data showed that a majority of CC1+ cells (red) were co-stained with Olig2 (green) throughout the brain (91.0% ~ 99.6%, white arrowheads, Fig. 3b, d-m) and spinal cord (98.70%, white arrowheads, Fig. 3n). On the other hand, while most brain regions showed high colocalization between Olig2 and CC1, we also found Olig2-positive but CC1-negative cells in some CNS regions (Fig. 3b-d, 3f-o, and Table 1). Particularly, in the striatum only 51.7% of Olig2-positive cells showed CC1 signal, suggesting that many Olig2+ cells are not oligodendrocytes in the striatum (Fig. 3e and Table 1). Together, these data suggest that there is a subpopulation of Olig2+ cells in some adult CNS regions that are neither oligodendrocytes nor OPCs.
Olig2 not detected in neurons and microglia
Neurons are electrically excitable cells that are the basic functional units of the CNS. Olig2 plays a crucial role in the generation of motor neurons in the developing spinal cord. We investigated whether Olig2 could be continuously expressed in some neurons of the adult CNS. However, we did not find any NeuN+ neurons that co-immunolabeled with Olig2 in either the brain or spinal cord (Fig. 4). Furthermore, we also performed co-immunostaining of Olig2 with microglia marker Iba1 and did not detect any Olig2+ cells co-expressing Iba1 (Fig. 5). These data indicate that both neurons and microglia do not express Olig2 in the adult mouse CNS.
Region-specific expression of Olig2 in astrocytes
Astrocytes are resident glial cells in the CNS besides oligodendrocytes and microglia. We further investigated whether Olig2 expression can be detected in the astrocytes of adult CNS by performing co-immunostaining of Olig2 with S100β, an astrocyte specific marker. As shown in the tiled Figure 6a, S100β signal was found throughout the adult CNS, with very high level immune reactivity detected in the OB, AON, MO, and the molecular layer of the cerebellum. Interestingly, high magnification confocal images found that many S100β+ astrocytes also expressed Olig2 in a region-specific pattern. In the AON, STR, HIP, CB (white arrows, Fig. 6c, e, i, l) and cortical areas including the MO, SS, VIS and the RSP (white arrows, Fig. 6d, f-h), very few Olig2 was located within astrocytes (quantified in Fig. 6p). Unexpectedly, we found a large number of S100β+ astrocytes that were also Olig2-positive in the OB, TH, MB, and MY (white arrowheads, Fig. 6b, j, k, m). Quantified data showed that the majority of astrocytes (>70%) in these regions co-expressed Olig2 (Fig. 6p). An even higher proportion of S100β+ astrocytes co-expressed Olig2 that were detected in the adult mouse spinal cord (89.3%, Fig. 6n-p). We also calculated the percentage of S100β+ and Olig2+ astrocytes among the total Olig2+ cells, and found that in some areas such as the OB, TH, MB, MY and SC, more than a quarter of Olig2+ cells are astrocytes (Table 1). Therefore, our results suggest that there is a distinct subpopulation of astrocytes with a high level of Olig2 expression in some specific regions in the adult mouse CNS.
To further confirm that astrocytes have Olig2 in some specific CNS regions, we performed co-immunostaining of Olig2 with another astrocytic marker Sox9. Similarly, we detected a significant number of Sox9+ cells that were also Olig2 positive in the TH, MB, MY and SC (white arrowheads, Supplementary Figure 1j, k, m and o). Quantified data showed that over half of Sox9+ cells also expressed Olig2 in the TH, MB, MY and SC. Especially, over 90% of Sox9+ cells in the spinal cord were also Olig2-expressing cells. Together, our results further confirm that astrocytes can express Olig2 in some regions of adult mouse brain and spinal cord.
Highest Olig2 expression level in OPCs
After discovering remarkable Olig2 expression in astrocytes within some specific regions, we further investigated whether the Olig2 expression level in astrocytes is comparable to that of OPCs and oligodendrocytes. When compared side-by-side among astrocytes, OPCs, and oligodendrocytes, we found that OPCs expressed much higher level of Olig2 signal than that of oligodendrocytes and astrocytes in the OB (Fig. 7a-c), and TH, MB, and MY, as well as the spinal cord (Fig. 7d, p<0.001). Interestingly, the Olig2 expression level was comparable between oligodendrocytes and astrocytes (Fig. 7). These results indicate that in some regions of the adult mouse CNS under normal conditions, astrocytes can express Olig2 at a similar level to the oligodendrocytes.