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First published online 11 April 2007
doi: 10.1242/dev.02847

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A crucial role for Olig2 in white matter astrocyte development

¹ Department of Developmental Biology and Kent Waldrep Foundation Center for Basic Neuroscience Research on Nerve Growth and Regeneration, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
² Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
³ Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
⁴ Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

View larger version (96K): [in this window] [in a new window]   Fig. 1. Transient Olig2 expression in developing cortical astrocytes. (A-E) Sagittal cortical sections from wild-type mice at P5 were immunostained with antibodies against GFAP, Olig2 or glutamine synthetase (GS). (A) GFAP+ cells (red) at this stage expressed a high level of Olig2 (green) as judged by staining intensity. Orthogonal reconstructions of confocal images at z-axis level are shown in side panels (along the right-hand edge and beneath). Arrowheads indicate GFAP+ Olig2+ double-positive cells. A GFAP+ Olig2+ cell, marked by cross-lines, is shown at a higher magnification in B-B''. (C-E) Co-expression (E) of GFAP (C, red) and GS (D, green) was observed in the astrocytes of the neonatal cortex. (F-K) Sagittal cortical sections of wild-type mice at P5 (F,G), P14 (H,I) and P21 (J,K) were immunostained with antibodies against Olig2 and GS. Confocal images at z-axis level are shown in side panels of G and I. (F,G) GS+ astrocytes (red) express a high level of Olig2 (green) at P5. Arrowheads indicate GS+ Olig2+ double-positive cells. (H,I) At P14, Olig2 expression in GS+ astrocytes (arrowheads) is reduced to a low level as compared with the intense Olig2 expression in GS- cells (blue arrows). (J,K) At P21, Olig2 expression (green) is undetectable in GS+ astrocytes (red) in the cortex. (L-O) The corpus callosum region of wild-type mice at P5 was immunostained with antibodies against GFAP and Olig2. Arrowheads indicate GFAP+ Olig2+ double-positive cells in this developing white matter. The boxed area in L is shown at a higher magnification in M-O. Orthogonal reconstructions of confocal images at z-axis level are shown for a GFAP+ Olig2+ cell marked by crosslines in O. Scale bars: 50 µm in A,F-L.

View larger version (78K): [in this window] [in a new window]   Fig. 2. Alteration of GFAP expression in the developing and adult Olig2-ablated cortex. Expression of Gfap was analyzed by in situ hybridization on coronal brain sections of control (Olig2C/+;hGFAPCre or CtrlG) and Olig2 mutant mice (Olig2Cko;hGFAPCre or CkoG) at P7 (A,B), P14 (C-F) and adult (P79; G-J). Neocortical Gfap expression is indicated by blue arrows. Red, blue and white boxed areas in C,D and G,H, representing the hippocampus and the neocortex from control and Olig2-ablated mice, are shown at high magnification in E,F, and I,J, respectively. Arrowheads and arrows in E,F indicate Gfap expression in the white matter tract and stratum lacunosum-moleculare of the hippocampus, respectively. Cortical lamination layers are indicated on the right-hand side of D.

View larger version (101K): [in this window] [in a new window]   Fig. 3. Astrocyte differentiation deficit in the white matter of the Olig2-ablated cortex. (A-D) Expression of GFAP (Red) and Olig2 (green) was analyzed by immunohistochemistry on coronal brain sections of control (A,C) and Olig2-ablated (B,D) mice at P14. Arrowheads indicate white matter. (E,F) Hematoxylin and Eosin (H/E) staining of sagittal sections of control (CtrlG) and Olig2-ablated (CkoG) cortices at P14. (G,H) Cortices of control (CtrlG) and Olig2 mutant (CkoG) mice at P14 were immunostained with an anti-S100ß antibody. Arrowheads in E-H indicate the corpus callosum (CC). (I-N) Double immunostaining of glutamine synthetase (GS, red) and GFAP (green) in the corpus callosum (arrowheads) of wild-type (I,J), CtrlG (K,L) and Olig2 mutant (CkoG; M,N) mice. GS+ and GS+ GFAP+ cells are shown in I,K,M and J,L,N, respectively. (O) Bar chart showing the average number of GS+ and S100ß+ cells per unit area (0.1 mm2) in the corpus callosum of wild-type (wt), CtrlG and Olig2 mutant (CkoG) mice (>300 cells counted, n=3); bars indicate s.d. Scale bars: 100 µm in A-D; 100 µm in G,H; 50 µm in I-N.

View larger version (88K): [in this window] [in a new window]   Fig. 4. Effects of neonatal Olig2 ablation on white matter astrocyte development. (A-D) Cre and GFAP expression in the cerebral white matter (A,B) and SVZ (C,D) of hGFAP-Cre mice at P14 was examined by immunohistochemistry. Arrowheads indicate Cre+ cells (B,D) and Cre+ GFAP+ cells (A,C). LV, lateral ventricle; wm, white matter. (E-L) Control (Olig2C/+;CAGGSCreErTM) and Olig2Cko;CAGGSCreErTM pups were administrated with tamoxifen (TM) for 4 days from P2 to P5, and their brains were harvested at P13. Expression of Olig2 (E,F), Mbp (G,H) and Gfap (I,J) was examined by in situ hybridization on coronal sections of the cortex. Boxed areas in I,J are shown at high magnification in K,L, respectively. Arrows and arrowheads indicate the white matter and the SVZ, respectively. (M) Bar chart showing the average number per unit area (0.1 mm2) of GS+ cells in the cortex (Ctx) and the white matter (Wm) and CC1+ cells in the white matter of TM-treated control (Olig2C/+;CAGGSCreErTM) and Olig2Cko;CAGGSCreErTM animals (n=3); bars indicate s.d. Scale bars: 100 µm in A for A,B, and C for C,D.

View larger version (104K): [in this window] [in a new window]   Fig. 5. White matter astrocyte formation deficiency in the Olig2 mutant spinal cord. In situ hybridization and immunostaining of Olig2, GFAP, S100ß and GS were performed on frozen spinal sections of CtrlG and CkoG mice at P14. Arrows indicate the white matter region of the spinal cord. (A,B) mRNA expression of Gfap was examined in the control (A) and Olig2 (B) mutant spinal cord by in situ hybridization. (C-F) Expression of Olig2 and GFAP was analyzed by immunohistochemistry in the control (C) and Olig2 mutant (E) spinal cord. GFAP expression in C,E is shown in D,F, respectively. GFAP is strongly expressed in the spinal white matter of the control, but only weakly in the mutant (arrows). (G-N) Expression of Olig2 and S100ß (G,I), and Olig2 and GS (K,M), were analyzed by immunohistochemistry in the spinal cord of control (G,H,K,L) and Olig2 mutant (I,J,M,N) mice. Staining of S100ß and GS is shown in H,J and L,N, respectively. S100ß- and GS-expressing cells are barely detectible in the white matter of Olig2 mutants (J,N) as compared with controls (H,L). (O) Bar chart showing the average number of S100ß+ and GS+ cells per unit area (0.06 mm2) in the spinal white matter of control and Olig2 mutant mice (n=3); bars indicate s.d. Scale bar: 100 µm in A-N.

View larger version (88K): [in this window] [in a new window]   Fig. 6. Formation of astrocytes with GFAP upregulation in the superficial layers of the Olig2-ablated cortex. (A,B) Hematoxylin and Eosin (H/E) staining of sagittal sections of control (CtrlG) and Olig2-ablated (CkoG) cortices at P14. Arrows indicate the superficial cortical layers. (C-F) Cortices of control and CkoG mice at P14 were immunolabeled with antibodies against GS and GFAP. Arrows indicate GS+ cells (C,D) and GS GFAP co-expressing cells (F). (G-I) Cortices of control (G,H) and CkoG (I) mice at P14 were immunostained with Olig2, S100ß and GFAP. Regions from superficial layers of the cortex are shown. Arrows in G indicate cells co-labeled with Olig2 and S100ß. Arrows in H,I indicate S100ß+ in the control and S100ß GFAP co-expressing cells in the Olig2 mutant, respectively. (J) Bar chart showing the average number of GS+ and S100ß+ cells per unit area (0.1 mm2) in the superficial layers of control and Olig2 mutant (Cko) mice (>400 cells counted, n=3); bars indicate s.d. (K-M) Cortices of control and CkoG animals at P14 were immunolabeled with GFAP (K-M), Ki67 (M) and BrdU (K,L) after a 4-hour pulse of BrdU administration before sacrifice. Arrows indicate BrdU+ and Ki67+ cells in the cortex, respectively. (N) Bar chart indicating the gross number of BrdU+ cells per field (0.4 mm2) in the superficial layers of control and Olig2- ablated (Cko) cortices (>200 cells counted, n=3) at P7 and P14; bars indicate s.d. (O,P) Immunostaining of Ki67 in the SVZ of the control (O) and Olig2 mutant (P) brains. Scale bars: 100 µm.

View larger version (92K): [in this window] [in a new window]   Fig. 7. Absence of ectopic GFAP expression in oligodendroglial precursors of the Olig2-ablated cortex. (A-B'') The Olig2 (CkoG) mutant mouse cortex at P14 was immunostained with GFAP and OPC markers NG2 (A-A'') and PDGFR (B-B''). Regions of the superficial layers of Olig2- ablated cortices are shown. Expression of GFAP is distinct from that of OPC markers. (C-F) Coronal sections of control (CtrlC) and Olig2Cko;CNPCre (CkoC) mutant cortices at P14 were subjected to in situ hybridization labeling for Mbp (C,D) and Gfap (E,F). Arrows indicate the cortical gray matter. (G,H) The corpus callosum of control (CtrlC) and Olig2 (CkoC) mutant cortices at P14 were immunostained with antibodies against GFAP (green) and GS (red).

View larger version (148K): [in this window] [in a new window]   Fig. 8. Absence of significant neuronal deficit in the Olig2-ablated cortex. (A-D) The cortices from control (CtrlG) and Olig2-ablated (CkoG) mice at P14 were immunostained with antibodies to NeuN (red), calbindin (red) and GABA (green), as indicated. (E,F) Gfap expression was examined by in situ hybridization in the forebrain of control Olig2C/+;Syn1Cre (CtrlS) and Olig2Cko;Syn1Cre (CkoS) mice. (G-J) Immunostaining of Olig2 (green) and axonal proteins NF200 (red) and Tau-1 (red) in the cortex of control (CtrlG; G,I) and Olig2 mutant (CkoG; H,J) mice. (K-N) Immunostaining of axonal proteins NF200 and Tau-1 in the corpus callosum (CC) of wild-type (K,M), and Olig2 mutant (CkoG; L,N) mice. The more intense staining in the CkoG mice, as compared with the wild-type mice, might be due to the fact that dysmyelinating axons in the Olig2 mutant are more accessible to the antibodies.

View larger version (77K): [in this window] [in a new window]   Fig. 9. A population of cortical astrocytes with GFAP upregulation is derived from Olig2-ablated cells. (A-D) Expression of Olig1 was examined by in situ hybridization in the coronal section of control and Olig2-ablated CkoG cortices at P8 (A,B) and P14 (C,D). Arrowheads above and below the dashed line in indicate the superficial layers and the white matter in the cortex, respectively. (E,F) In situ hybridization for Olig1 and Gfap in the gray matter. Arrows above and below the longer dashed line indicate the superficial and deep cortical layers, respectively. (G) Schematic showing fate-mapping of Olig2-ablated cells after hGFAPCre-mediated recombination. Breeding of hGFAP-Cre mice with Olig2-floxed mice carrying the RosaYFP reporter gene (Srinivas et al., 2001) generated offspring with Olig2 ablation and activation of YFP expression in the same cells after Cre-mediated excision of loxP sites (black triangles). Red line indicates Olig2-3' UTR. (H) Immunostaining with antibodies to YFP and GFAP in the superficial cortical layers of Olig2-ablated triple-transgenic mice at P14. Arrowheads indicate cells co-labeled with YFP and GFAP. (I,J) Expression of Olig2-3' UTR and Gfap was examined by double in situ hybridization on sections of the Olig2-ablated cortex at P14. Arrows in I indicate Olig2-expressing cells in the deep cortical layers. Boxed area in I is shown at high magnification in J. Arrowheads in J indicate cells co-expressing Olig2-3' UTR and Gfap. (K) Schematic depicting cortical astrocyte development in relation to Olig2 expression. In the normal developing brain, Olig2 is expressed in cortical neural progenitor cells (NP). At early postnatal stages, Olig2 is expressed in immature developing astrocytes (A) at a high level. Beginning at postnatal week 2, the Olig2 expression level is reduced in astrocytes and GFAP expression becomes undetectable. In adulthood, Olig2 expression is absent in mature astrocytes, suggesting that the Olig2 expression level is downregulated in cortical astrocytes during the process of astrocyte maturation in brain development. GFAP downregulation in mature astrocytes suggests that other factors also negatively regulate GFAP expression in adulthood. In the absence of Olig2, GFAP expression is sustained or upregulated in a population of cortical astrocytes throughout adulthood. By contrast, Olig2 ablation results in a defect in the formation of white matter astrocyte subpopulations (lower panel).

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