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The Journal of Neuroscience, July 2, 2003, 23(13):5561-5571
Previous Article | Next Article
The Role of ErbB2 Signaling in the Onset of Terminal Differentiation of Oligodendrocytes In Vivo
Ju Young Kim,1,2 Qin Sun,1 Michael Oglesbee,3 andSung Ok Yoon1 1Neurobiotechnology Center and Department of Molecular and Cellular Biochemistry, 2Molecular, Cellular, and Developmental Biology Program, and 3Department of Veterinary Biosciences, Ohio State University, Columbus, Ohio 43210
Abstract
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Abstract
Introduction
The knock-out analyses of neuregulin and its receptors have indicated that they play essential roles in Schwann cell development. However, the role they play in oligodendrocyte development in vivo has remained unclear, because such knock-out animals die before CNS myelination begins. We examined the role of neuregulin signaling in the CNS by generating transgenic mice that express a dominant-negative mutant of the ErbB2 receptor among oligodendrocytes, using an MBP promoter. The transgenic mice exhibited widespread hypomyelination, resulting from a reduction in oligodendrocyte differentiation. The number of progenitors was conversely increased in the transgenic mice. We report that a reduction in oligodendrocyte differentiation is attributed in part to apoptosis of oligodendrocyte progenitors as they exit the cell cycle. A significant reduction in the number of p27+ oligodendrocyte precursors in the transgenic mice supports this conclusion. Taken together, these data suggest that for oligodendrocyte progenitors, ErbB2 signaling plays a role in governing a properly timed exit from the cell cycle during development into myelinating oligodendrocytes.
Key words: ErbB2; neuregulin; p27; cell-cycle exit; differentiation; oligodendrocytes
Introduction
The development of oligodendrocytes is regulated by both cell intrinsic factors as well as cell extrinsic factors. Cell intrinsic factors include oligodendrogenic genes, olig-1, olig-2, and Sox10 (Lu et al., 2002; Stolt et al., 2002
and NG2 (chondroitin sulfate proteoglycan) in vivo (Levine and Stallcup, 1987
It has been shown that axons or axon-derived factors play essential roles for different aspects of oligodendrocyte development, such as proliferation, differentiation, and survival, in addition to myelination (Barres and Raff, 1999
In this report, we sought to examine the role of NRG/ErbB2 in vivo, by perturbing the signaling ability of ErbB2 among only oligodendrocytes, through the expression of a dominant-negative ErbB2 receptor under MBP promoter in transgenic mice. Although knock-out mice have been generated to analyze the function of NRG signaling, clarification of its role in vivo has remained untenable, because knock-out mice of NRG-1 and their receptors, ErbB2, ErbB3, and ErbB4, die before myelination begins in the CNS, mainly of heart defects (Gassmann et al., 1995
Materials and Methods
Generation of transgenic constructs and identification of transgenic mice: MBP-p75–ErbB1KD. The cDNA for the p75–ErbB1 KD construct has been described previously (Harrington et al., 2002-globin gene, and (2) ligating the p75–ErbB1 KD fragment and the flanking sequences containing the exon 2–intron 2 of the
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Figure 2. The expression of the p75–ErbB1 KD follows the pattern of endogenous MBP expression during development. A, A diagram of the transgenic p75–ErbB1 KD construct under the MBP promoter. The location of PCR primers that were used for identification of transgene (TG) is indicated by arrows. The angled arrow indicates the start of transcription. Translation starts at the beginning of the transgene. B, The expression of the p75–ErbB1 KD during development. MBP staining was first observed at P5 in the white-matter tracts of the cerebellum; it increased as development proceeded. The p75–ErbB1 KD expression was followed by HA staining in transgenic littermates. Note the reduction in MBP staining in TG compared with NTG at each developmental time point analyzed. Scale bar, 20 µm. C, The p75–ErbB1 KD is targeted to the cell surface in culture. Mouse oligodendrocytes were stained live with anti-HA to detect the p75–ErbB1 KD on the cell surface. The NTG cultures do not show HA staining. Mouse oligodendrocyte cultures were as described previously (Harrington et al., 2002
EM analyses. For transmission electron microscopy (TEM) of the tissues, mice were perfused with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M Na cacodylate buffer, pH 7.2. The tissues were dissected, rinsed in 0.1 M Na cacodylate buffer, and placed in 1% osmium and 0.1 M Na cacodylate for 1–1.5 hr at room temperature. The tissues were stained en bloc for 1 hr in 2% uranyl acetate and embedded in Spurr resin after the dehydration procedures. Sections were cut at 80 nm using an Ultracut E ultramicrotome (Leica, Deerfield, IL), and collected on 300-mesh grids. Sections were stained in 2% uranyl acetate and Reynolds lead citrate before observation in a Philips (Wilmington, DE) CM 12 TEM at 60 kV.
Immunohistochemistry. Mice were perfused with 3% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.2. The brain was dissected, postfixed for several hours at room temperature, and placed in 20% sucrose and 0.1 M PB at 4°C. The brain was cut at 20 µm thicknesses on a cryostat in the sagittal plane. For staining with antibodies, the sections were incubated in blocking solution containing 1% BSA, 0.3% Triton X-100, and 10% horse serum for mouse monoclonal antibody or 10% goat serum for rabbit polyclonal antibody in 0.1 M PB for 2 hr at room temperature. The primary antibodies were incubated in 5% horse or goat serum, 0.1% BSA in 0.1 M PB overnight at room temperature in the presence of 0.02% sodium azide. The positive staining was detected using Alexa-488- or Cy3-conjugated secondary antibodies, or biotinylated secondary antibodies that were later visualized using streptavidin-Cy3 or Alexa-488. The sections were mounted using Vectashield containing 4',6'-diamidino-2-phenylindole to stain the nuclei (Vector Laboratories, Burlingame, CA). For staining with anti-bromodeoxyuridine (BrdU), mice were injected with BrdU at 25 µg/g three times at 2 hr intervals. Two hours after the last injection, mice were perfused. BrdU staining was detected essentially as described previously (Seri et al., 2001
Immunoprecipitation/Western blot analyses. Mouse brains were homogenized in a lysis buffer containing 1% Nonidet P-40, 20 mM Tris, pH 8.0, 137 mM NaCl, 0.5 mM EDTA, 10% glycerol, 10 mM Na2P2O7, 10mM NaF, 1 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM vanadate, and 1 mM phenylmethylsulfonyl fluoride. The procedures for immunoprecipitation and Western blotting were essentially as described previously (Yoon et al., 1998
Receptor autokinase assays. The ErbB2 receptors in the mouse brain lysates were immunoprecipitated using antibodies to ErbB2 (Santa Cruz Biotechnology), and the ErbB2 receptor immune complexes were washed in HNTG buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 10 mM NaF, 1 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM vanadate, and 1 mM phenylmethylsulfonyl fluoride. The receptor kinase reaction was performed in HNTG buffer plus 5 mM MnCl2, 20 µM ATP, and 20 µCi of [
-32P]ATP at 4°C for 15 min as described previously (Pinkas-Kramarski et al., 1996
Quantitation of CC1+/TUNEL+, CC1+, and BrdU+ cells. Every fourth sagittal section (20 µm in thickness) was processed for immunohistochemistry; positively stained cells were counted from the cerebellar white matter.
Mouse oligodendrocyte cultures. The procedures were performed as described previously (Harrington et al., 2002
Results
The p75–ErbB1KD construct is described in Figure 1A. The construct comprises two domains: one containing the cytoplasmic domain of ErbB1 with its ATP binding site inactivated, and the other containing the extracellular and transmembrane domains of a neurotrophin receptor, p75. The rationale behind choosing the p75–ErbB1KD as a dominant-negative mutant for ErbB2 is that ErbB1 is a preferred heterodimeric partner for ErbB2 with high affinity, in response to the widest range of ligands in the NRG family (Jones et al., 1999
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Figure 1. The p75–ErbB1 KD associates with ErbB2 and inhibits NRG-dependent activation of ErbB2 in 293 cells. A, Experimental strategy of the p75–ErbB1 KD construct. The p75–ErbB1 KD would heterodimerize with ErbB receptors and inhibit tyrosine phosphorylation of the associated ErbB receptors. The p75–ErbB1 KD itself does not bind EGF, because the ligand-binding domain is replaced with the corresponding domain of p75. B, The p75–ErbB1 KD does not affect TrkA signaling in the presence of NGF. 293T cells were transfected with TrkA, and subsequently infected with the control GFP or p75–ErbB1 KD adenovirus. After 5 min of treatment with NGF, the lysates were immunoprecipitated with pan-Trk antibody, and the activation status of TrkA was detected using 4G10 antibody. The same blot was reprobed for the presence of the transfected TrkA using pan-Trk antibody in IP/W. Note that tyrosine phosphorylation of TrkA is not affected in the presence of the p75–ErbB1 KD. The presence of the p75–ErbB1 KD is shown as an HA blot. C, The p75–ErbB1 KD associates only with ErbB2, but not with ErbB3 or ErbB4 in 293T cells. 293T cells were transfected with ErbB2, 3, or 4, and subsequently infected with the control GFP or p75–ErbB1 KD adenovirus. After immunoprecipitation with antibodies against ErbB2, 3, or 4, or HA tag included in the p75–ErbB1 KD, the associated p75–ErbB1 KD was detected with anti-HA antibody. A portion of the lysates was subjected in parallel to IP/W, using ErbB2, 3, or 4 antibodies as controls. D, The p75–ErbB1 KD inhibits NRG-dependent activation of ErbB2. 293T cells were transfected with ErbB4 to facilitate activation of the endogenous ErbB2 with NRG, and subsequently infected with the control GFP or p75–ErbB1 KD adenovirus. After 5 min of treatment of soluble NRG, the lysates were immunoprecipitated with ErbB2 antibody and the activation status of ErbB2 was detected using PY99 antibody. The presence of the p75–ErbB1 KD was detected with HA antibody in IP/W. Note that tyrosine phosphorylation of ErbB4 is also reduced in the presence of the p75–ErbB1 KD. E, ErbB4 is present in the p75–ErbB1 KD–ErbB2 complex. 293T cells were transfected with Flag–ErbB4 to facilitate activation of the endogenous ErbB2 with NRG, and subsequently infected with the control GFP or p75–ErbB1 KD adenovirus. After 5 min of treatment of soluble NRG, the lysates were immunoprecipitated with ErbB2 antibody, and the presence of ErbB4 and the p75–ErbB1 KD was probed with anti-Flag or HA antibodies. W:PY, Phosphotyrosine.
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Figure 3. ErbB1 and p75 are not expressed among oligodendrocyte lineage. A, ErbB1 is not expressed in oligodendrocyte lineage. Sagittal brain sections from P5 and P25 mice were analyzed for ErbB1 expression among CC1 + cells. Staining with TG sections is shown as a positive control, because ErbB1 antibody detects the cytoplasmic domain of the human ErbB1 that is present in the p75–ErbB1 KD. At P5, there is some positive staining for ErbB1 in the cerebellar white matter, but it did not colocalize with CC1. These stainings represent migrating cerebellar granule neurons. In TG, subpopulations of CC1 + cells begin to stain for ErbB1 at P5 (arrows). At P25, there is no ErbB1 staining in the cerebellar white matter, nor is there HA staining. In TG at P25, however, almost every cell body positive for ErbB1 is also stained for HA (arrow). B, p75 is not expressed in oligodendrocyte lineage. Sagittal brain sections from P5 and P25 rats were analyzed for p75 expression among oligodendrocytes using CC1 and NG2 as oligodendrocyte markers. At P5 there were many p75 +, CC1 +, and NG2 + cells in the cerebellar white matter, but p75 staining did not colocalize with CC1 or NG2. At P25 there was no p75 staining detected in the cerebellar white matter. Scale bars, 12.5 µm.
The ability of the p75–ErbB1KD to function as a dominant-negative mutant to ErbB2 was characterized in 293T cells. To test its association with ErbB receptors, 293T cells were transfected with ErbB2, 3, or 4 and subsequently infected with either the GFP control or the p75–ErbB1KD adenovirus. After immunoprecipitation with antibodies against ErbB2, 3, or 4, the p75–ErbB1KD was found to be in a complex with ErbB2, but not ErbB3 or 4 (Fig. 1C). We next tested whether the association with the p75–ErbB1KD results in modulation of ErbB2 signaling in the presence of NRG. For this, 293T cells were transfected with ErbB4 to activate endogenous ErbB2 in 293T cells, because ErbB2 does not bind NRG directly. Twenty-four hours after infection with the p75–ErbB1KD or GFP adenoviruses, cells were treated for 10 min with 25 ng/ml of soluble NRG. The extent of ErbB2 activation was measured in immunoprecipitation/Western blot analyses (IP/W), using ErbB2 antibody for the immunoprecipitation and phosphotyrosine antibodies for the Western blot. In control cells, NRG induced tyrosine phosphorylation of ErbB2 and ErbB4 (Fig. 1D). However, in the presence of the p75–ErbB1KD there was significant attenuation of the NRG-mediated tyrosine phosphorylation of ErbB2. These results indicate that the p75–ErbB1KD can function as a dominant-negative mutant for the endogenous ErbB2, a major signaling receptor in NRG signaling (Graus-Porta et al., 1997
To assess the role of NRG–ErbB2 signaling in oligodendrocyte development in vivo, we introduced the p75–ErbB1KD under MBP promoter (Fig. 2A). The MBP promoter contains 9 kb of 5' sequence as described previously (Forghani et al., 2001
We first examined whether the expression of the p75–ErbB1KD followed the expression pattern of endogenous MBP. Sagittal brain sections from the transgenic mice (TG) were stained for HA to detect the presence of the p75–ErbB1KD (Fig. 2B), whereas the brains from both TG and nontransgenic control (NTG) littermates were analyzed for endogenous MBP expression at P5, P10, P15, P20, and P25 (Fig. 2B). For the analyses, we focused on the white-matter tract in the cerebellum. The level of the endogenous MBP was weak at P5, and its expression increased gradually, developing into an adult pattern of white-matter MBP expression at P25. The p75–ErbB1KD expression pattern was also detected weakly at P5 based on HA staining, with a gradual increase into adulthood. This result suggests that the p75–ErbB1KD expression followed the endogenous MBP expression pattern in a correct temporal manner. Using a 3.2 kb MBP promoter linked to LacZ, Foran and Peterson (1992
To ensure that there is no cross talk between the p75–ErbB1KD and ErbB1 or p75 in vivo, we examined whether oligodendrocytes express p75 or ErbB1 during development in the CNS. For endogenous ErbB1 expression, we stained sagittal brain sections from P5 and P25 with ErbB1 antibody and CC1, a marker for oligodendrocytes. In our study, CC1 staining is almost identical to GalC staining, a postmitotic marker for oligodendrocytes (Richardson et al., 1988
The expression of p75 during oligodendrocyte development was examined using rat brain sections, because of antibody compatibility. In the white-matter tract of the cerebellum at P5, there were many p75+ cells, but its staining did not colocalize with either CC1 or NG2 (Fig. 3B). The cells that are p75+ at that time represent migrating cerebellar granule cells. At P25 there was no p75 staining detected in the cerebellar white matter, although many cells were positive for CC1. Therefore, these data indicate that p75 is not expressed in oligodendrocyte lineage during normal development, although injury can induce p75 expression among oligodendrocytes in the CNS or culturing itself can induce p75 (Beattie et al., 2002
The behavioral phenotype and the MBP expression level in our transgenic mice indicated a defect in myelination. Therefore, we processed the adult spinal cord of the two transgenic lines for the electron microscopy (EM) analyses. In both transgenic lines, there was a reduction in myelinated fibers in the ventral funiculi (Fig. 4A). The extent of hypomyelination was first quantified by counting the number of axon fibers that were myelinated regardless of thickness using the TG-2 line. In NTG, 70% of fibers within all funiculi were myelinated and 30% were unmyelinated. In TG this was reversed, with 35% of fibers myelinated and 65% unmyelinated (Fig. 4B). Although thickness was not considered when making the counts, it should be noted that even among the myelinated fibers, the myelin sheath is much thinner in TG than in NTG. Therefore, we evaluated the ratio of myelin thickness to axon diameter. In NTG, the myelin thickness increased as the axon diameter increased, but in TG-2, the increase in the axon diameter was not accompanied in a similar proportion (Fig. 4C). Based on MBP staining of sagittal brain sections, the hypomyelination phenotype was widespread in the CNS. An example is shown in Figure 4D, with a focus on the corticospinal tract and the cerebellum (see also Fig. 2B). It should be noted in Figure 4D that reduced neurofilament staining accompanied the reduction in MBP staining, confirming a tight relationship between axons and oligodendrocyte development as proposed previously (Barres and Raff, 1999
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Figure 4. Widespread hypomyelination in the p75–ErbB1 KD mice. A, Representative EM pictures from two transgenic lines and a littermate control. Scale bar, 400 nm. B, Quantification of hypomyelination phenotype in the spinal cord of TG-2 line. The ratio of myelinated to unmyelinated axon fibers is reversed from 7 to 3 in NTG to 3.5 to 6.5 in TG-2. For quantification of myelinated axons from the ventral funiculus of the lumbar spinal cord, two rows of four grids (eight grids total) were prepared from tissues adjacent to the ventral medial fissure for the generation of electron photomicrographs. The data are from three sets of NTG and TG mice at P15. Note that the myelin sheath is also thinner among the myelinated fibers in TG. C, Relationship between the myelin thickness and axon diameter of the myelinated axons. The data are from 30 randomly selected myelinated axons from each group. D, The extent of hypomyelination in the cerebellum (Cb) and in the corticospinal tract (CST). Sagittal sections from P25 mice were processed for double immunohistochemistry with MBP and neurofilament (NF) antibodies. Scale bar, 12.5 µm.
We next examined whether the hypomyelination phenotype was attributable to perturbation of ErbB2 signaling in vivo. The p75–ErbB1KD was present in immune complexes of ErbB2, but not of ErbB3 or ErbB4, when the brain and spinal cord lysates were analyzed (Fig. 5A, top). This result suggests that the p75–ErbB1KD interacts with only ErbB2 in vivo, as in 293T cells. The total levels of these three receptors were very similar when comparing TG with NTG (Fig. 5A, bottom), suggesting that the expression of the p75–ErbB1KD did not affect the overall level of these receptors. The consequence of this physical association between the p75–ErbB1KD and the endogenous ErbB2 was examined. For this, ErbB2 was immunoprecipitated, and the ability of ErbB2 to phosphorylate its oligomeric partners was assessed by adding 32P-
45% reduction in the amount of radiolabeled ErbB2 compared with its littermate control (Fig. 5B). A similar reduction in tyrosine phosphorylation of ErbB2 was also observed in TG compared with controls (data not shown). These data indicate that ErbB2 signaling was significantly attenuated in vivo in the presence of the p75–ErbB1KD.
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Figure 5. Attenuation of ErbB2 signaling by the p75–ErbB1KD in vivo. A, The p75–ErbB1KD associates with the endogenous ErbB2, but not with ErbB3 or ErbB4 in vivo. The brain and the spinal cord lysates from adult mice were subjected to IP using ErbB2, 3, or 4 or HA antibodies followed by Western blotting with anti-HA to detect associated p75–ErbB1KD. Note that the level of three ErbB receptors did not change in the presence of the p75–ErbB1KD. B, The p75–ErbB1KD inhibits the signaling ability of the endogenous ErbB2 by 45%. The brain lysates were subjected to receptor autokinase assays using 32P-
When the p75–ErbB1KD associates with ErbB2, ErbB2 can potentially phosphorylate the p75–ErbB1KD at tyrosine residues. This could lead to the recruitment of ErbB1 adaptor molecules and subsequent signaling events. To determine whether the cytoplasmic ErbB1KD domain in the p75–ErbB1KD is phosphorylated at tyrosine residues on association with ErbB2, we immunoprecipitated ErbB2 and blotted with anti-phosphoErB1. There was no tyrosine phosphorylation in the ErbB1KD domain that associated with ErbB2, in two independent TG lines (Fig. 5C, top). Because the phosphoErbB1 antibody detects only the phosphorylation status of tyrosine 845, it is possible that other tyrosine residues may become phosphorylated by association. For this, we subjected the ErbB2 immune complexes to an autokinase assay to detect the overall phosphorylation status of the p75–ErbB1KD, as was done in Figure 5B. Even with increasing amounts of lysates, there was no phosphorylation of the p75–ErbB1KD (Fig. 5C, bottom, arrow). These data suggest that the cytoplasmic ErbB1KD domain in the p75–ErbB1KD is not phosphorylated by the endogenous ErbB2. This lack of cross-phosphorylation may suggest that ligand binding is necessary for the proper conformational change and subsequent autophosphorylation between receptor tyrosine kinases.
We next examined the outcome of attenuated ErbB2 signaling in oligodendrocyte development. Brain sections taken from different developmental time points were stained with differentiation markers, such as O1, and CNPase. MBP staining is shown in Figure 2B. The data with O1 staining in the white-matter tract of the cerebellum is shown in Figure 6A, but similar data were obtained with CNPase as well as MBP staining throughout the CNS (corpus callosum, corticospinal tract, brainstem, spinal cord). O1 staining increased as development proceeded both in TG and NTG, but at each time point, there was a significant reduction in O1 staining in TG compared with NTG. By adulthood, the number of mature oligodendrocytes was reduced by 47% in TG compared with NTG (Fig. 6B). This reduction in oligodendrocyte differentiation could result from a decrease in the number of progenitors. Therefore, we assessed the extent of proliferation using BrdU staining. In the white-matter tract of the cerebellum in both NTG and TG, the number of BrdU+ cells decreased as development proceeded (Fig. 6C,D). However, at each developmental stage analyzed, the number of BrdU+ cells in TG was higher than that in NTG. Similarly, the number of cells that were positive for both NG2, a marker for oligodendrocyte progenitors (Levine and Stallcup, 1987
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Figure 6. A subpopulation of postmitotic oligodendrocytes undergoes apoptosis. A, Reduction in oligodendrocyte differentiation in the p75–ErbB1 KD mice. Sagittal brain sections were stained for O1 at different developmental stages. O1 staining was reduced in the white-matter tracts of the cerebellum in the transgenic mice compared with NTG at every developmental stage analyzed. A similar pattern was also observed with CNPase staining (data not shown). Scale bar, 20 µm. B, Reduction in the number of mature oligodendrocytes in the p75–ErbB1 KD mice. Quantification of CC1 + cells was done from the white-matter tract of the cerebellum using P25 mice (n = 3). Error bars indicate SEM. C, An increase in BrdU incorporation in the p75–ErbB1 KD. Sagittal brain sections were stained with anti-BrdU. BrdU staining was increased in the white-matter tracts of the cerebellum in the p75–ErbB1 KD mice compared with NTG at every developmental stage analyzed. Scale bar, 20 µm. D, Quantification of BrdU + cells in the cerebellar white-matter tract during development. Error bars indicate SEM. E, Increased proliferation of NG2 + cells in the transgenic mice. The P5 brain sections were stained for NG2 and PCNA. Note the increase in the number of NG2 +/PCNA + cells in the white-matter tracts in the cerebellum (arrows). Scale bar, 12.5 µm.
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Figure 7. An increase in apoptosis of oligodendrocytes in the p75–ErbB1 KD mice. A, Quantification of TUNEL + cells at P5 (n = 3). Representative pictures from the cerebellum white matter (right). Error bars indicate SEM. B, Quantification of TUNEL + cells among CC1 + cells at P5(n=3). For quantification, see Materials and Methods. Representative pictures from the cerebellar white matter (right). An arrow points to TUNEL +CC1 + cells that were counted. Scale bar, 12.5 µm.
The level of p27, a cell cycle inhibitor, has been shown to increase as oligodendrocytes differentiate in culture (Durand et al., 1997
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Figure 8. P27 level is significantly decreased in the p75–ErbB1 KD mice. A, Quantification of p27 in Western blot analyses. Actin blot was used as a control. B, The spinal cord sections taken from P10 mice were stained for CC1 and p27 or CNPase and p27. Left, Low-magnification pictures; scale bar, 20µm. Middle and right, High-magnification pictures; scale bar, 8.3µm. Note that some cells in the p75–ErbB1 KD mice have p27 in the nucleus, but its level is much lower than that found in NTG (arrows). C, The p75–ErbB1 KD is expressed among some dividing oligodendrocyte progenitors. The p75–ErbB1 KD was detected by ErbB1 staining in the cerebellar white matter at P5 and P10. Note that the cells that are positive for ErbB1 contain BrdU immunoreactivity in their nucleus.
Discussion
In this report, we provide data showing that NRG signaling plays critical, early roles in oligodendrocyte differentiation during development in vivo. With a 45% reduction in ErbB2 signaling, there was a 47% decrease in the number of mature oligodendrocytes in the adult. This reduction in myelinating oligodendrocytes did not stem from a reduction in oligodendrocyte progenitors, but from the apoptotic death of cells that appeared to have failed to exit the cell cycle at appropriate times. This conclusion is based on our data showing that there was an increase in the proliferation of NG2+ oligodendrocyte progenitors and a significant reduction in p27 level in the transgenic mice with attenuated ErbB2 signaling. Taken together, these data suggest that NRG signaling via ErbB2 plays a role in regulating cell cycle exit timing during oligodendrocyte development.Vartanian et al. (1994
It should be noted that in our transgenic mice, ErbB2 signaling is perturbed only after oligodendrocytes begin to express MBP. Our approach is similar to the Krox-20-cre-mediated conditional deletion of ErbB2 among committed, promyelinating, and myelinating Schwann cells (Garratt et al., 2000
Because the perturbation of ErbB2 signaling coincided with oligodendrocyte differentiation in our transgenic mice, we cannot completely rule out the possibility that ErbB2 signaling also plays a role in the proliferation of oligodendrocyte progenitors in vivo. This case appears especially plausible, considering that in Schwann cells, NRG/ErbB2 provides both a mitogenic signal as well as cues critical for myelination (Morris et al., 1999
The p75–ErbB1KD construct can bind neurotrophins via the extracellular and transmembrane domains of p75 (Harrington et al., 2002
In conclusion, we report that ErbB2 signaling plays a critical role in oligodendrocyte differentiation in vivo, in part by regulating the onset of terminal differentiation in oligodendrocyte development. Perturbation of such ErbB2 signaling leads to a widespread hypomyelination in the CNS.
Footnotes
Received Jan. 2, 2003; revised Apr. 2, 2003; accepted Apr. 4, 2003.This work was supported by grants from the American Cancer Society and the Whitehall Foundation, and by National Institutes of Health Grant RO1 NS39472-01 (S.O.Y.). We thank Dr. Alexander Gow for the pMG2 construct; Dr. Alan Peterson for the 9 kb MBP promoter constructs; Dr. Nancy Hynes for ErbB2, ErbB3, and ErbB4 constructs; Dr. Lin Mei for Flag-ErbB4 construct; Kathy Wolken for the EM analyses; Dr. Jan Parker-Thornberg for generating transgenic mice; and Drs. Doug L. Falls, Kuo-Fen Lee, Pilar Perez, and Bruce Carter for critical comments of this manuscript.
Correspondence should be addressed to Dr. Sung Ok Yoon, The Neurobiotechnology Center and Department of Molecular and Cellular Biochemistry, 184 Rightmire Hall, 1060 Carmack Road, Ohio State University, Columbus, OH 43210. E-mail: yoon.84{at}osu.edu.
Copyright © 2003 Society for Neuroscience 0270-6474/03/235561-11$15.00/0
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