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Populations of radial glial cells respond differently to reelin and neuregulin1 in a ferret model of cortical dysplasia.

Poluch S, Juliano SL - PLoS ONE (2010)

Bottom Line: Ferrets treated with MAM on E24 result in an overall decrease of BLBP expression; radial glia that continue to express BLBP, however, show only mild disruption compared with the strongly disrupted vimentin expressing radial glia.We further investigated the effects induced by reelin and found that signaling was mediated via VLDLR/Dab1/Pi3K activation while NRG1 signaling was mediated via erbB3/erbB4/Pi3K.We then tested whether radial glial repair correlated with improved neuronal migration.

View Article: PubMed Central - PubMed

Affiliation: Anatomy, Physiology, and Genetics, Uniformed Services University, Bethesda, Maryland, United States of America.

ABSTRACT
Radial glial cells play an essential role during corticogenesis through their function as neural precursors and guides of neuronal migration. Both reelin and neuregulin1 (NRG1) maintain the radial glial scaffold; they also induce expression of Brain Lipid Binding Protein (BLBP), a well known marker of radial glia. Although radial glia in normal ferrets express both vimentin and BLBP, this coexpression diverges at P3; vimentin is expressed in the radial glial processes, while BLBP appears in cells detached from the ventricular zone. Our lab developed a model of cortical dysplasia in the ferret, resulting in impaired migration of neurons into the cortical plate and disordered radial glia. This occurs after exposure to the antimitotic methylazoxymethanol (MAM) on the 24th day of development (E24). Ferrets treated with MAM on E24 result in an overall decrease of BLBP expression; radial glia that continue to express BLBP, however, show only mild disruption compared with the strongly disrupted vimentin expressing radial glia. When E24 MAM-treated organotypic slices are exposed to reelin or NRG1, the severely disrupted vimentin+ radial glial processes are repaired but the slightly disordered BLBP+ processes are not. The realignment of vimentin+ processes was linked with an increase of their BLBP expression. BLBP expressing radial glia are distinguished by being both less affected by MAM treatment and by attempts at repair. We further investigated the effects induced by reelin and found that signaling was mediated via VLDLR/Dab1/Pi3K activation while NRG1 signaling was mediated via erbB3/erbB4/Pi3K. We then tested whether radial glial repair correlated with improved neuronal migration. Repairing the radial glial scaffold is not sufficient to restore neuronal migration; although reelin improves migration of neurons toward the cortical plate signaling through ApoER2/Dab1/PI3K activation, NRG1 does not.

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Deviation of vimentin+ and BLBP+ fibers in E24 MAM treated organotypic cultures exposed to variant forms of NRG1.(A–G) Immunostaining against vimentin (red) and BLBP (green) after 2 DIC. (A, n = 6), depicts control slices incubated in plain medium. Vimentin+ radial glia realign when MAM treated slices are incubated with 1 nM of recombinant NRG1 (B, n = 4) or cocultured with Ig-NRG1 cells (C, n = 4). The morphology of vimentin+ radial glia was not improved in cocultures with CRD-NRG1 cells (D, n = 4). The effect of recombinant NRG1 was abolished in presence of antibodies blocking erbB3 (20 µg/ml) (E, n = 5) or erbB4 (20 µg/ml) (F, n = 6), and in presence of a Pi3K inhibitor LY294002 (50 µM) (G, n = 7). (See Figure S1). (a) illustrates slices in A–B,E–F cultured in plain medium or medium supplemented with drugs. Slices in C and D were cocultured with HEK cells as shown in (b). (H) Histogram illustrating the degrees of deviation for vimentin+ and BLBP+ radial glial processes. (I) Histogram of the percentage of processes expressing vimentin and BLBP (vim+BLBP+, orange), only vimentin (vim+BLBP-, red) or only BLPB (BLBP+ vim-, green). n =  number of slices. Error bars  =  standard error. Significance was determined using a Two-way ANOVA followed by pairwise multiple comparison procedures (Holm-Sidak method). **p<0.001, *p = 0.003. Scale Bar: 25 µm.
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pone-0013709-g006: Deviation of vimentin+ and BLBP+ fibers in E24 MAM treated organotypic cultures exposed to variant forms of NRG1.(A–G) Immunostaining against vimentin (red) and BLBP (green) after 2 DIC. (A, n = 6), depicts control slices incubated in plain medium. Vimentin+ radial glia realign when MAM treated slices are incubated with 1 nM of recombinant NRG1 (B, n = 4) or cocultured with Ig-NRG1 cells (C, n = 4). The morphology of vimentin+ radial glia was not improved in cocultures with CRD-NRG1 cells (D, n = 4). The effect of recombinant NRG1 was abolished in presence of antibodies blocking erbB3 (20 µg/ml) (E, n = 5) or erbB4 (20 µg/ml) (F, n = 6), and in presence of a Pi3K inhibitor LY294002 (50 µM) (G, n = 7). (See Figure S1). (a) illustrates slices in A–B,E–F cultured in plain medium or medium supplemented with drugs. Slices in C and D were cocultured with HEK cells as shown in (b). (H) Histogram illustrating the degrees of deviation for vimentin+ and BLBP+ radial glial processes. (I) Histogram of the percentage of processes expressing vimentin and BLBP (vim+BLBP+, orange), only vimentin (vim+BLBP-, red) or only BLPB (BLBP+ vim-, green). n =  number of slices. Error bars  =  standard error. Significance was determined using a Two-way ANOVA followed by pairwise multiple comparison procedures (Holm-Sidak method). **p<0.001, *p = 0.003. Scale Bar: 25 µm.

Mentions: Soluble recombinant type I NRG1 repairs the radial glial scaffold in E24 MAM ferret slices [28]. To assess whether NRG1 acts similarly on both vimentin+ and BLBP+ radial glia, organotypic slices were exposed for 2 DIC to NRG1, and the degrees of deviation computed for vimentin+ and BLBP+ radial glial processes (see Table 1). MAM treated slices were initially exposed to a diffuse source of recombinant NRG1 (1 nM) as described previously [28]. We observed a dramatic realignment of vimentin+ radial glia with an angle of deviation at 10.43, which is significantly reduced from the angle of deviation of vimentin+ radial glia in MAM treated slices incubated 2 DIC in plain medium (19.21) (Figure 6A,B,H). As described above using reelin, BLBP+ radial glial morphology, although much less disrupted in MAM treated ferrets compared to vimentin+ radial glia, was unchanged after treatment with recombinant NRG1 (9.22 compared to 10.76 in plain medium; not significant, p>0.05). Again, the morphology of BLBP+ radial glia was similar to the improved vimentin+ processes. The soluble recombinant NRG1 used here is a truncated form of NRG1, which contains only the soluble EGF-like domain. This form of NRG1, commonly used for in vitro studies, is sufficient to elicit ErbB receptor dimerization, tyrosine phosphorylation and the activation of downstream signaling pathways [51]. We additionally decided to expose MAM treated slices to the full length of NRG1 to assess if other domains beyond the EGF-like domain could improve the BLBP+ radial glial morphology. E24 MAM slices were co-cultured with HEK cells secreting the full length of type I NRG1 (Ig-NRG1 cells). HEK cells were included in Matrigel and placed at the pial surface as described previously. The morphology of vimentin+ radial glial cells was dramatically improved, comparable to a treatment with soluble recombinant NRG1 (degree of deviation of 11.08; Figure 6C,H). However, BLBP+ radial glia remained the same in the presence of the full length of NRG1 (degree of deviation of 9.72), but similar to the improved vimentin+ radial glia. Finally, we co-cultured MAM slices with HEK cells expressing type III NRG1 (CRD-NRG1 cells). The isoform III, unlike the Ig-like domain of type I NRG1, is not secreted and contains a cysteine-rich domain (CRD). We found no improvement of the radial glial scaffold: with a degree of deviation of 20.51 for vimentin and 10.91 for BLBP, suggesting that the morphology of radial glia in presence of type III NRG1 was similar to MAM slices cultured in plain medium (Figure 6D,H). Therefore, the soluble EGF-like domain of type I NRG1, applied diffusely or focally, is sufficient to realign vimentin+ radial glia, which are highly disrupted in MAM treated animals; BLBP+ radial glia, although less disrupted, remain unchanged.


Populations of radial glial cells respond differently to reelin and neuregulin1 in a ferret model of cortical dysplasia.

Poluch S, Juliano SL - PLoS ONE (2010)

Deviation of vimentin+ and BLBP+ fibers in E24 MAM treated organotypic cultures exposed to variant forms of NRG1.(A–G) Immunostaining against vimentin (red) and BLBP (green) after 2 DIC. (A, n = 6), depicts control slices incubated in plain medium. Vimentin+ radial glia realign when MAM treated slices are incubated with 1 nM of recombinant NRG1 (B, n = 4) or cocultured with Ig-NRG1 cells (C, n = 4). The morphology of vimentin+ radial glia was not improved in cocultures with CRD-NRG1 cells (D, n = 4). The effect of recombinant NRG1 was abolished in presence of antibodies blocking erbB3 (20 µg/ml) (E, n = 5) or erbB4 (20 µg/ml) (F, n = 6), and in presence of a Pi3K inhibitor LY294002 (50 µM) (G, n = 7). (See Figure S1). (a) illustrates slices in A–B,E–F cultured in plain medium or medium supplemented with drugs. Slices in C and D were cocultured with HEK cells as shown in (b). (H) Histogram illustrating the degrees of deviation for vimentin+ and BLBP+ radial glial processes. (I) Histogram of the percentage of processes expressing vimentin and BLBP (vim+BLBP+, orange), only vimentin (vim+BLBP-, red) or only BLPB (BLBP+ vim-, green). n =  number of slices. Error bars  =  standard error. Significance was determined using a Two-way ANOVA followed by pairwise multiple comparison procedures (Holm-Sidak method). **p<0.001, *p = 0.003. Scale Bar: 25 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965671&req=5

pone-0013709-g006: Deviation of vimentin+ and BLBP+ fibers in E24 MAM treated organotypic cultures exposed to variant forms of NRG1.(A–G) Immunostaining against vimentin (red) and BLBP (green) after 2 DIC. (A, n = 6), depicts control slices incubated in plain medium. Vimentin+ radial glia realign when MAM treated slices are incubated with 1 nM of recombinant NRG1 (B, n = 4) or cocultured with Ig-NRG1 cells (C, n = 4). The morphology of vimentin+ radial glia was not improved in cocultures with CRD-NRG1 cells (D, n = 4). The effect of recombinant NRG1 was abolished in presence of antibodies blocking erbB3 (20 µg/ml) (E, n = 5) or erbB4 (20 µg/ml) (F, n = 6), and in presence of a Pi3K inhibitor LY294002 (50 µM) (G, n = 7). (See Figure S1). (a) illustrates slices in A–B,E–F cultured in plain medium or medium supplemented with drugs. Slices in C and D were cocultured with HEK cells as shown in (b). (H) Histogram illustrating the degrees of deviation for vimentin+ and BLBP+ radial glial processes. (I) Histogram of the percentage of processes expressing vimentin and BLBP (vim+BLBP+, orange), only vimentin (vim+BLBP-, red) or only BLPB (BLBP+ vim-, green). n =  number of slices. Error bars  =  standard error. Significance was determined using a Two-way ANOVA followed by pairwise multiple comparison procedures (Holm-Sidak method). **p<0.001, *p = 0.003. Scale Bar: 25 µm.
Mentions: Soluble recombinant type I NRG1 repairs the radial glial scaffold in E24 MAM ferret slices [28]. To assess whether NRG1 acts similarly on both vimentin+ and BLBP+ radial glia, organotypic slices were exposed for 2 DIC to NRG1, and the degrees of deviation computed for vimentin+ and BLBP+ radial glial processes (see Table 1). MAM treated slices were initially exposed to a diffuse source of recombinant NRG1 (1 nM) as described previously [28]. We observed a dramatic realignment of vimentin+ radial glia with an angle of deviation at 10.43, which is significantly reduced from the angle of deviation of vimentin+ radial glia in MAM treated slices incubated 2 DIC in plain medium (19.21) (Figure 6A,B,H). As described above using reelin, BLBP+ radial glial morphology, although much less disrupted in MAM treated ferrets compared to vimentin+ radial glia, was unchanged after treatment with recombinant NRG1 (9.22 compared to 10.76 in plain medium; not significant, p>0.05). Again, the morphology of BLBP+ radial glia was similar to the improved vimentin+ processes. The soluble recombinant NRG1 used here is a truncated form of NRG1, which contains only the soluble EGF-like domain. This form of NRG1, commonly used for in vitro studies, is sufficient to elicit ErbB receptor dimerization, tyrosine phosphorylation and the activation of downstream signaling pathways [51]. We additionally decided to expose MAM treated slices to the full length of NRG1 to assess if other domains beyond the EGF-like domain could improve the BLBP+ radial glial morphology. E24 MAM slices were co-cultured with HEK cells secreting the full length of type I NRG1 (Ig-NRG1 cells). HEK cells were included in Matrigel and placed at the pial surface as described previously. The morphology of vimentin+ radial glial cells was dramatically improved, comparable to a treatment with soluble recombinant NRG1 (degree of deviation of 11.08; Figure 6C,H). However, BLBP+ radial glia remained the same in the presence of the full length of NRG1 (degree of deviation of 9.72), but similar to the improved vimentin+ radial glia. Finally, we co-cultured MAM slices with HEK cells expressing type III NRG1 (CRD-NRG1 cells). The isoform III, unlike the Ig-like domain of type I NRG1, is not secreted and contains a cysteine-rich domain (CRD). We found no improvement of the radial glial scaffold: with a degree of deviation of 20.51 for vimentin and 10.91 for BLBP, suggesting that the morphology of radial glia in presence of type III NRG1 was similar to MAM slices cultured in plain medium (Figure 6D,H). Therefore, the soluble EGF-like domain of type I NRG1, applied diffusely or focally, is sufficient to realign vimentin+ radial glia, which are highly disrupted in MAM treated animals; BLBP+ radial glia, although less disrupted, remain unchanged.

Bottom Line: Ferrets treated with MAM on E24 result in an overall decrease of BLBP expression; radial glia that continue to express BLBP, however, show only mild disruption compared with the strongly disrupted vimentin expressing radial glia.We further investigated the effects induced by reelin and found that signaling was mediated via VLDLR/Dab1/Pi3K activation while NRG1 signaling was mediated via erbB3/erbB4/Pi3K.We then tested whether radial glial repair correlated with improved neuronal migration.

View Article: PubMed Central - PubMed

Affiliation: Anatomy, Physiology, and Genetics, Uniformed Services University, Bethesda, Maryland, United States of America.

ABSTRACT
Radial glial cells play an essential role during corticogenesis through their function as neural precursors and guides of neuronal migration. Both reelin and neuregulin1 (NRG1) maintain the radial glial scaffold; they also induce expression of Brain Lipid Binding Protein (BLBP), a well known marker of radial glia. Although radial glia in normal ferrets express both vimentin and BLBP, this coexpression diverges at P3; vimentin is expressed in the radial glial processes, while BLBP appears in cells detached from the ventricular zone. Our lab developed a model of cortical dysplasia in the ferret, resulting in impaired migration of neurons into the cortical plate and disordered radial glia. This occurs after exposure to the antimitotic methylazoxymethanol (MAM) on the 24th day of development (E24). Ferrets treated with MAM on E24 result in an overall decrease of BLBP expression; radial glia that continue to express BLBP, however, show only mild disruption compared with the strongly disrupted vimentin expressing radial glia. When E24 MAM-treated organotypic slices are exposed to reelin or NRG1, the severely disrupted vimentin+ radial glial processes are repaired but the slightly disordered BLBP+ processes are not. The realignment of vimentin+ processes was linked with an increase of their BLBP expression. BLBP expressing radial glia are distinguished by being both less affected by MAM treatment and by attempts at repair. We further investigated the effects induced by reelin and found that signaling was mediated via VLDLR/Dab1/Pi3K activation while NRG1 signaling was mediated via erbB3/erbB4/Pi3K. We then tested whether radial glial repair correlated with improved neuronal migration. Repairing the radial glial scaffold is not sufficient to restore neuronal migration; although reelin improves migration of neurons toward the cortical plate signaling through ApoER2/Dab1/PI3K activation, NRG1 does not.

Show MeSH
Related in: MedlinePlus