1. Volume 25, Issue 2, Pages 317-329 (February 2000)
Sonic Hedgehog–Regulated Oligodendrocyte Lineage Genes Encoding bHLH Proteins in the Mammalian Central Nervous System 
Q.Richard Lu, Dong-in Yuk, John A Alberta, Zhimin Zhu, Inka Pawlitzky, Joanne Chan, Andrew P McMahon, Charles D Stiles, David H Rowitch 
Neuron 
Volume 25, Issue 2, Pages (February 2000)
DOI: /S (00)

2. Figure 1
Predicted Primary Structure, Homology, and Expression of Olg mRNA Transcripts
(A) Amino acid sequence of rat Olg-1 protein is depicted, showing both the bHLH (underline) and serine-theronine-rich (boxed) domains in the N-terminal region.
(B) Alignment of the bHLH domains of rat Olg-1/2 and the bHLH transcription factors NDRF, NeuroD, and Ngn2.
(C) Cartoon illustrating structural features of rat and human Olg genes, showing the conserved bHLH domain (80% identity) and serine-theronine-rich domain.
(D) Olg-1/2 is expressed exclusively in the brain of adult rats. A commercial Northern blot (Clonetech) of mRNA extracted from various tissues of the adult rat was probed with 32P-labeled Olg-1 or Olg-2, revealing 2.5 and 2.7 kb mRNA transcripts in brain.
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3. Figure 2 Olg-1/2 Expression in Oligodendrocytes of the Adult Rat CNS
Transverse sections of adult rat cerebral cortex and cerebellum and logitudinal sections of optic nerves were submitted to in situ hybridization and immunohistochemistry.
(A) Low-power (2×) view of brain showing strong expression of Olg-1 in the corpus callosum (cc) and hemispheric white matter.
(B) The corpus callosum at higher magnification (20×) reveals that Olg-1-positive cells have round nuclei and are grouped in linear arrays, indicated as arrows, which are characteristic features of oligodendrocytes.
(C) Similar result in the corpus callosum is obtained for Olg-2.
(D) Region with expression of Olg-1 in the corpus callosum corresponds to that highly labeled with the oligodendrocyte marker O4.
(E and F) Olg-1-expressing cells in the hippocampus of adult rat in distribution similar to that of the oligodendrocyte marker O4.
(G and H) Expression of Olg-1 is confined to cerebellar white matter (cwm), which contains oligodendrocytes labeled by O4. Olg-1 expression is never found in the internal granular (igl) or molecular (mol) layers.
(I) Olg-1 expression by in situ hybridization on a longitudinal section of optic nerve from P14 rat reveals linear arrays of cell bodies (arrows), a characteristic of oligodendrocytes (20×).
(J) A similar result in optic nerve is obtained for Olg-2 (20×).
(K) Cells with APC antibody (CC-1) immunostaining with DAB substrate (visualized as brown color) show linear arrays of oligodendrocytes in P14 optic nerve (20×).
(L) Cells with APC antibody immunostaining (brown color) after in situ hybridization for Olg-1 (blue color) show that the majority of Olg-1-expressing cells are also colabeled by APC antibody (CC-1; dark blue color, indicated by arrows) in P14 optic nerve (20×). Cells with Olg-1 expression, but not APC antibody staining, were also found, as indicated by arrowhead.
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4. Figure 3
Colocalization of Olg-1-Positive Cells with Oligodendrocyte Marker Protein APC, but Not Astrocyte Marker S100β, in Optic Nerve from P14 Rat
Double immunostains using an Olg-1 peptide antibody were performed in longitudinal optic nerve sections at 10–20 μm. Left column: top, CC-1 antibody (Cy2, green) immunolabels cells that form as linear arrays (arrows); middle, Olg-1 antibody (Cy3, red) also immunostains cells in linear arrays (arrows); bottom, overlay image shows colocalization of cells that express APC and Olg-1. Right column: top, S100β antibody immunostain (Cy2, green); middle, Olg-1 antibody immunostain (Cy3, red); bottom, overlay image shows that S100β and Olg-1 are expressed in a mutually exclusive pattern. Small areas of color overlay probably reflect overlapping cells within these 10–20 μm tissue sections.
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5. Figure 4
Expression of Olg-1/2 in the Embryonic CNS Precedes PDGFRα and DM20/PLP
(A–D) Lateral views of mouse embryos subjected to whole-mount in situ hybridization.
(A) A 9.0 dpc embryo. Olg-1 mRNA transcripts were detected in the ventral spinal cord (closed arrows), with rostral restriction approximately at the second rhombomere of the hindbrain (r2) and in the ventral diencephalon (open arrow).
(B and C) At 10.5 dpc, Olg-1 and Olg-2 expression persisted in the spinal cord (closed arrows). Note decreasing caudal-to-rostral intensity of signal. Expression was also detected at the prosomere 2/3 boundary (open arrow) separating the telencephalon (tel) from the diencephalon (di), and in the nasal placode (np) and optic (ov) and otic (ot) vesicles.
(D) A 10.5 dpc cross-section of Olg-2 whole-mount in situ hybridization shows ventral spinal cord localization (arrows). Section shown corresponds to dashed line shown in (C).
(E) A 10.5 dpc embryo showing DM20/PLP expression in the mes-diencephalon (open arrow). Note that DM20/PLP was not detected in the spinal cord at 10.5 dpc, but strong signal is detected in the dorsal root ganglia (drg; closed arrow).
(F–M) In situ hybridization on transverse sections from 12.5 dpc (F–I) and 14.5 dpc (J–M) mouse embryos.
(F and G) Olg-1/2 expression was largely confined to the ventral ventricular region and was absent in the dorsal root ganglia.
(H) Onset of PDGFRα expression in the ventral spinal cord (arrow).
(I) DM20/PLP expression is persistent in the dorsal root ganglia (arrow) but is not expressed in the spinal cord at 12.5 dpc.
(J–M) By 14.5 dpc, expression of Olg-1/2, PDGFRα, and DM20/PLP occurs in a similar distribution of cells apparently migrating from ventral to dorsal regions of the spinal cord. Note that some nonadjacent sections are shown, which may account for differences in pattern observed in the panels from similar time points.
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6. Figure 5
Regional Expression of Olg-1 in Ventricular Zone Precursor Cells Overlaps Expression of the Early Oligodendrocyte Marker PDGFRα
Transverse sections of 13.5 dpc mouse embryonic ventral spinal cord (cartoon to left shows region of analysis), which was subjected to in situ hybridization and photographed at 40x.
(A) Expression of Shh, which marks the floor plate (indicated by dashed lines).
(B) Nkx-2.2 is regionally expressed (arrow indicates dorsal border) adjacent to floor plate.
(C) Expression of Pax6 occurs in a region dorsal to that of Nkx-2.2. Note a region of “high” Pax6 expression (inferior border indicated by arrow), dorsal to a region of relatively low Pax6 expression in the ventricular zone (Ericson et al. 1997b).
(D) Olg-1 mRNA transcripts were detected in a region (indicated by arrows) dorsal to Nkx-2.2-expressing cells in a region of low Pax6 expression.
(E) PDGFRα is expressed in a domain similar to that of Olg-1.
(F) Summary of results indicating the position of Olg-1-expressing cells relative to the neuronal precursor markers Pax6 and Nkx-2.2 (right-hand side; Tanabe and Jessell 1996; Ericson et al. 1997a; Briscoe et al. 1999). The left side shows the approximate locations of somatic motor neurons (MNs). Figure adapted from Briscoe et al
(G) Double labeling of Olg-1- (purple color) and PDGFRα- (brown color) expressing cells in E16 rat embryonic spinal cord by in situ hybridization. Numerous cells that colabeled were identified, some of which are indicated (closed arrows). Cells that only labeled for PDGFRα expression were also seen (open arrow). The floor plate (fp) and ventricular zone (vz) are indicated.
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7. Figure 6
Infection of Embryonic Cortical Explants with an Olg-1-Carrying Virus Results in Formation of NG2-Positive Oligodendroglial Precursor Cells
E13.5 rat cortical precursor cell explants were infected either with control adenovirus AdeGFP (A–C) or with the Olg-1 cDNA–carrying adenovirus AdeGFP-Olg-1 (D–F). After 48 hr in culture, samples were immunostained with antibody against NG2 and subsequently visualized either by fluorescence microscopy for GFP (A and D) or NG2 (B and E) or by phase microscopy (C and F). Arrows indicate AdeGFP-Olg-1-infected cells within the same field (E and F).
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8. Figure 7
Ectopic Olg-1 Expression and Oligodendrocyte Precursor Cell Development in Transgenic Mice that Ectopically Express Shh
In situ hybridization was performed on transverse sections of 14.5 dpc spinal cord from Shh-Tg mice in which Shh is ectopically expressed in the dorsal CNS under control of the Wnt-1 regulatory element (Rowitch et al. 1999).
(A) Low-power (4×) view.
(B) High power (20×) view showing that dorsal expression of Shh is confined largely to greatly enlarged ventricular zones (vz). Normal expression of Shh in the floor plate (fp) is indicated.
(C and D) Striking induction of Olg-1 is observed in regions of the ectopic ventricular zone in Shh-Tg mice, including cells lining the lumen.
(E and F) In regions adjacent to the ectopic ventricular zone, induction of PDGFRα expression is observed.
(G) In contrast to the results above, DM20/PLP expression is not ectopically induced.
(H) Identification of oligodendrocyte precursor cells by the marker O4 in the dorsal CNS of 17.5 dpc Shh-Tg mice. Note that O4-positive cells (open arrows) are found adjacent to ectopic ventricular zone, identified by dense staining of cell nuclei with DAPI (left panel, 20× magnification; right panel, 100×).
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9. Figure 8
Upregulation of Olg-1 in 14.5 dpc Rat Cortical Neuroepithelial Cells by Shh Protein, as Indicated by Northern Blot Analysis
(A) Rapid upregulation of Olg-1 in neuroepithelial explant cultures treated (for 3 hr) with 250 ng/ml (13 nM) of N-terminal 19K fragment of mouse N-Shh. Total RNA (10 μg/lane) was analyzed; expression of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is shown as a control for loading.
(B) Bell-shaped dose–response curve for upregulation of Olg-1 by N-Shh protein. Neuroepithelial explant cultures were exposed for 3 hr to the indicated amounts of N-Shh. The bar graph (upper) depicts composite results of two to three separate experiments at each indicated dose. One representative experiment is shown as an inset (lower).
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10. Figure 9
Shh Is Required for Normal Expression of Olg-1 and Olg-2 in the CNS
Embryos homozygous for null mutations of Shh and wild-type or heterozygous litter mates were harvested at 10.5 dpc and submitted to whole-mount in situ hybridization.
(A) Note Shh expression in the floor plate (fp) at spinal cord levels. In the brain, Shh is expressed in the ventral diencephalon (di) and telencephalon (t) and in the zli (Echelard et al. 1993; Marti et al. 1995).
(B and C) Expression of Olg-1 in wild-type (B) and Shh mutant (C) embryos.
(C) Note that normal expression of Olg-1 in the spinal cord and brain is absent, while otic vesicle (ot) mRNA transcripts are detected.
(D and E) Expression of Olg-2 in wild-type (D) and Shh mutant (E) embryos.
(D) Olg-2 is expressed in the spinal cord in a decreasing caudal-to-rostral pattern. Olg-2 spinal cord expression terminates rostrally approximately at the second rhombomere of the hindbrain (r2). In addition, Olg-2 mRNA transcripts are detected at the zli, infundibulum, ventral telencephalon (t), and nasal placode (np).
(E) Note that normal expression of Olg-2 in the spinal cord and brain is absent, while nasal placode expression is maintained. Staining in the telencephalic vesicle is sporadic and most likely background.
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