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First published online 28 November 2007
doi: 10.1242/dev.004895

Development 135, 145-157 (2008)
Published by The Company of Biologists 2008
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NG2 cells generate both oligodendrocytes and gray matter astrocytes

Xiaoqin Zhu1, Dwight E. Bergles2 and Akiko Nishiyama1,*

1 Department of Physiology and Neurobiology, University of Connecticut, 75 North Eagleville Road, Storrs, CT 06269-3156, USA.
2 Department of Neuroscience, Johns Hopkins University School of Medicine, 813 Wood Basic Science Building, 725 N. Wolfe Street, Baltimore, MD 21205, USA.


Figure 1
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  		Fig. 1. Distribution of DsRed fluorescence and NG2 immunoreactivity in P3 and P30 tg cerebral cortex, cerebellum and spinal cord. (A,D) Coronal sections through P3 (A) and P30 (D) cerebral cortex. (B,E) Sagittal sections through P3 (B) and P30 (E) cerebellum. (C,F) Transverse sections through P3 (C) and P30 (F) spinal cord. Vibratome sections (100 µm) were immunolabeled with anti-NG2 antibodies and Alexa Fluor 488 goat anti-rabbit immunoglobulins. DsRed fluorescence (red) is localized to the somata of NG2-immunoreactive (green) cells. DsRed+NG2+ cells are distributed uniformly throughout the CNS. Scale bars: 20 µm.

 

Figure 2
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  		Fig. 2. Cellular localization of DsRed in coronal sections of P30 tg brain. (A,B) DsRed fluorescence (red) is detected in all the cells that are NG2+ (green) in corpus callosum (A) and cerebral cortex (B). (C) All the cells exhibiting DsRed fluorescence in the cerebral cortex express PDGFR{alpha}+ (green). (D) Comparison of DsRed fluorescence (red) and DsRed protein detected by a rabbit anti-DsRed antibody (green) in the cerebral cortex. DsRed protein is detected in the soma and throughout the processes. (E) Comparison of DsRed fluorescence with the oligodendrocyte antigen APC (green) in the corpus callosum. Strongly DsRed+ cells are negative for APC (arrows), whereas some weakly DsRed+ cells are APC+ (arrowheads). (F,G) DsRed fluorescence is not detected in astrocytes expressing GFAP (F) or S100β (G) in the cerebral cortex. (H) DsRed fluorescence is not detected in F4/80+ microglia in the cerebral cortex. (I) DsRed fluorescence is not detected in NeuN+ neurons in the cerebral cortex. Yellow indicates regions of contact, but the two antigens are not present in the same cells. (J,K) DsRed fluorescence is detected in PDGFRβ+ pericytes (K, arrowheads) but not in CD31+ endothelial cells (J, arrowheads) in the cerebral cortex. Scale bars: 12.5 µm in A-C; 20 µm in D,E; 25 µm in F-K.

 

Figure 3
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  		Fig. 3. Cre expression in NG2creBAC:Z/EG double tg mice. Coronal sections (50 µm) through dorsal and ventral cortex and corpus callosum from a P14 double tg mouse were double labeled with rabbit anti-Cre (red in B,D,F,H,J,L) and guinea pig anti-NG2 (blue in C,D,G,H,K,L) antibodies. (A-D) In dorsal cortex (DC) Cre is expressed in NG2+ cells with EGFP fluorescence (arrows). (E-H) In ventral cortex (VC) Cre is expressed in NG2+ cells with EGFP fluorescence (arrowheads), but not in EGFP+ protoplasmic astrocytes, which are devoid of NG2 (arrows). (I-L) In the corpus callosum (CC) Cre is expressed in NG2+ cells with EGFP fluorescence (arrowheads), but not in EGFP+NG2- oligodendrocytes with large cell bodies and few short and thick processes (arrows). Cre is only present in NG2+ cells and not in EGFP-expressing astrocytes or oligodendrocytes in NG2creBAC:Z/EG double tg mouse. Scale bars: 20 µm.

 

Figure 4
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  		Fig. 4. EGFP is expressed in APC+ oligodendrocytes and myelin in the white matter of P14 NG2CreBAC:Z/EG double tg mouse. Coronal sections (50 µm) through corpus callosum or striatum from P14 double tg mouse were double labeled with rabbit anti-EGFP (A,D,G; green), and mouse anti-APC (B,E,H; red) antibodies. (A-C) Projected images of z-stacks from the corpus callosum. The distribution of EGFP+ cells is similar to that of APC+ cells with extensive overlap between EGFP and APC. (D-I) Higher magnification of projected images of z-stacks from the corpus callosum (D-F) and striatum (G-I). APC+ oligodendrocytes in the corpus callosum express EGFP (straight arrows). EGFP-expressing NG2 glia are negative for APC (arrowheads). Some of the APC+ oligodendrocytes do not express EGFP (wavy arrows). EGFP+ oligodendrocytes form myelin, which is also EGFP+ (asterisks). Scale bars: 20 µm.

 

Figure 5
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  		Fig. 5. EGFP+ astrocytes in ventral but not dorsal cortex of P14 NG2CreBAC:Z/EG tg mice. Coronal sections (50 µm) through dorsal and ventral cortex and corpus callosum from P14 double tg mice were double or triple labeled with rabbit anti-EGFP (green in A,C,D,F,G,J,K,N,O,Q), mouse anti-GFAP (red in B,C,P,Q), mouse anti-S100β (red in E,F; blue in I,J,M,N), and guinea pig anti-NG2 (red in H,J,L,N) antibodies. (A-F) High magnification of EGFP+ astrocytes in ventral cortex. EGFP+ cells that have the typical morphology of protoplasmic astrocytes with bushy processes are immunoreactive for GFAP (A-C) or S100β (D-F). (G-J) EGFP+ cells in dorsal cortex are immunoreactive for NG2. S100β+ protoplasmic astrocytes in dorsal cortex are negative for NG2 and EGFP. (K-N) EGFP+ cells in ventral cortex extending bushy processes characteristic of protoplasmic astrocytes are also immunoreactive for S100β (thin arrows in M and N). EGFP+ NG2 cells are positive for NG2 but devoid of S100β (thick arrows). Some S100β+NG2- cells do not express EGFP (arrowheads). (O-Q) None of the EGFP+ cells in the corpus callosum express GFAP. Yellow areas represent overlap of GFAP and APC from adjacent cells, but the two antigens are never present in the same cells. Scale bars: 20 µm.

 

Figure 6
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  		Fig. 6. NG2 cell-astrocyte transition in P1 NG2creBAC:Z/EG double tg mice. (A-D) Coronal sections (50 µm) through ventral cortex from a P1 double tg mouse were triple labeled with rabbit anti-EGFP (green in A,D), guinea pig anti-NG2 (red in B,D) and mouse anti-nestin (blue in C,D) antibodies. An EGFP+nestin+ cell (straight arrows) with protoplasmic astrocytic morphology and weak NG2 expression extends a process with a broad end-foot (wavy arrows) that contacts a blood vessel (asterisks). Two typical NG2 glia on the right with strong NG2 expression (arrowheads) do not have vascular end-feet. Scale bars: 20 µm.

 

Figure 7
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  		Fig. 7. Distribution of EGFP+ cells in embryonic forebrain of NG2creBAC:Z/EG double tg mice. Coronal sections (20 µm) through the forebrain of an E18 double tg mouse were triple labeled with mouse anti-GFP (green in B,C,E,F), rabbit anti-NG2 (blue in B,C,E,F), and rabbit anti-GLAST (red in B,C,D,E,F) antibodies. Dorsal is top, medial is left in B and right in C-F. (A) The drawings represent E18 posterior (left) and anterior (right) forebrains. The boxed areas indicate locations of the images in B-F. LV, lateral ventricle. (B) A coronal section through the ventral subpial region of anterior forebrain. Pial surface is lower left. The majority of EGFP+ cells in anterior ventral forebrain express NG2 (arrows). A small number of EGFP+ cells express GLAST and are NG2- (arrowhead). (C) Ventral ventricular zone of posterior forebrain showing the ventral tip of the posterior horn of the lateral ventricle. EGFP is not expressed in GLAST+ radial glia extending from the ventral germinal zone. EGFP and NG2 expression in the SVZ is restricted to vascular cells. (D) A higher magnification of the image shown in C stained for GLAST and DAPI. The cells bodies of radial glia in the ventricular zone are labeled by GLAST and are negative for GFP. Strong EGFP expression seen in the lateral ventricle in C and D is in the choroid plexus. (E) A region immediately ventral to the ventral tip of the SVZ underlying the ventral VZ. All the EGFP+ cells in this region express NG2. GLAST+ radial glial fibers are EGFP-negative. EGFP+NG2+ cells in the parenchyma have long slender processes. (F) A region ventral to E, adjacent to the ventral pial surface. Clusters of EGFP+ cells localized close to the ventral pial surface consist of two cell types: EGFP+NG2+ cells with long slender processes (arrows) and EGFP+GLAST+ cells (arrowheads) with little or no NG2. Some of the latter EGFP+GLAST+ cells have bushy processes resembling immature protoplasmic astrocytes (cell with a star, shown in green channel in inset). Scale bars: 20 µm.

 

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