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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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Position of BrdU+ cells in E24 MAM treated organotypic cultures exposed to variant form of NRG1.After a pulse of BrdU, MAM treated slices were incubated 2 days in culture (DIC) in plain medium (A, n = 6) or in a medium containing 1 nM (B, n = 5) or 30 nM (C, n = 4) of recombinant NRG1 as shown in (a). Some slices were also cocultured with Ig-NRG1 HEK cells (D, n = 7) or CRD-NRG1 HEK cells (E, n = 6) as shown in (b). High magnification of BrdU+ cells in the cortical plate (CP) after 2 DIC in plain medium (A') or in presence of 1 nM NRG1 (B'). The positions of BrdU+ cells in 3 cortical compartments, CP, upper and lower intermediate zone (IZU and IZL) was analyzed after 2 DIC as shown in (c) (nuclear staining) and (d) (BrdU immunostaining of a slice cultured in plain medium). (J) Histogram of the position of BrdU+ cells. No significant differences were found between control (plain medium) and different forms of NRG1; in all conditions, BrdU+ cells distribute in a typical pattern for an E24 MAM treated slice in that they are spread throughout cortical wall. n =  number of slices. Error bars  =  standard error. Two-way ANOVA followed by pairwise multiple comparison procedures (Holm-Sidak method). Scale Bar: 150 µm.
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pone-0013709-g007: Position of BrdU+ cells in E24 MAM treated organotypic cultures exposed to variant form of NRG1.After a pulse of BrdU, MAM treated slices were incubated 2 days in culture (DIC) in plain medium (A, n = 6) or in a medium containing 1 nM (B, n = 5) or 30 nM (C, n = 4) of recombinant NRG1 as shown in (a). Some slices were also cocultured with Ig-NRG1 HEK cells (D, n = 7) or CRD-NRG1 HEK cells (E, n = 6) as shown in (b). High magnification of BrdU+ cells in the cortical plate (CP) after 2 DIC in plain medium (A') or in presence of 1 nM NRG1 (B'). The positions of BrdU+ cells in 3 cortical compartments, CP, upper and lower intermediate zone (IZU and IZL) was analyzed after 2 DIC as shown in (c) (nuclear staining) and (d) (BrdU immunostaining of a slice cultured in plain medium). (J) Histogram of the position of BrdU+ cells. No significant differences were found between control (plain medium) and different forms of NRG1; in all conditions, BrdU+ cells distribute in a typical pattern for an E24 MAM treated slice in that they are spread throughout cortical wall. n =  number of slices. Error bars  =  standard error. Two-way ANOVA followed by pairwise multiple comparison procedures (Holm-Sidak method). Scale Bar: 150 µm.

Mentions: To determine the ability of cells to migrate in organotypic cultures of either normal, E24 MAM treated cortex alone, or after coculture with HEK 293T cells embedded in Matrigel (as describe above), we plotted the distribution of BrdU+ cells after 2 DIC. Boundaries were drawn indicating the pia of a ferret slice and the outer edge of the VZ. This region was divided into 3 equal bins for each coculture and the number of cells per bin counted in a slab 500 µm in width in the somatosensory cortex. The bins correspond to the intermediate zone close to the VZ (i.e., the lowest part of the intermediate zone, IZL), a region in the IZ, but closer to the cortical plate (i.e., the upper part of the intermediate zone, IZU), and the region corresponding to the cortical plate (CP) (Figure 5c–d and Figure 7c–d). Histograms were made to indicate the position of BrdU+ cells across animals in each condition. To compare across samples, the number of cells/bin were calculated as a percent of the total number of cells in each slice.


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)

Position of BrdU+ cells in E24 MAM treated organotypic cultures exposed to variant form of NRG1.After a pulse of BrdU, MAM treated slices were incubated 2 days in culture (DIC) in plain medium (A, n = 6) or in a medium containing 1 nM (B, n = 5) or 30 nM (C, n = 4) of recombinant NRG1 as shown in (a). Some slices were also cocultured with Ig-NRG1 HEK cells (D, n = 7) or CRD-NRG1 HEK cells (E, n = 6) as shown in (b). High magnification of BrdU+ cells in the cortical plate (CP) after 2 DIC in plain medium (A') or in presence of 1 nM NRG1 (B'). The positions of BrdU+ cells in 3 cortical compartments, CP, upper and lower intermediate zone (IZU and IZL) was analyzed after 2 DIC as shown in (c) (nuclear staining) and (d) (BrdU immunostaining of a slice cultured in plain medium). (J) Histogram of the position of BrdU+ cells. No significant differences were found between control (plain medium) and different forms of NRG1; in all conditions, BrdU+ cells distribute in a typical pattern for an E24 MAM treated slice in that they are spread throughout cortical wall. n =  number of slices. Error bars  =  standard error. Two-way ANOVA followed by pairwise multiple comparison procedures (Holm-Sidak method). Scale Bar: 150 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965671&req=5

pone-0013709-g007: Position of BrdU+ cells in E24 MAM treated organotypic cultures exposed to variant form of NRG1.After a pulse of BrdU, MAM treated slices were incubated 2 days in culture (DIC) in plain medium (A, n = 6) or in a medium containing 1 nM (B, n = 5) or 30 nM (C, n = 4) of recombinant NRG1 as shown in (a). Some slices were also cocultured with Ig-NRG1 HEK cells (D, n = 7) or CRD-NRG1 HEK cells (E, n = 6) as shown in (b). High magnification of BrdU+ cells in the cortical plate (CP) after 2 DIC in plain medium (A') or in presence of 1 nM NRG1 (B'). The positions of BrdU+ cells in 3 cortical compartments, CP, upper and lower intermediate zone (IZU and IZL) was analyzed after 2 DIC as shown in (c) (nuclear staining) and (d) (BrdU immunostaining of a slice cultured in plain medium). (J) Histogram of the position of BrdU+ cells. No significant differences were found between control (plain medium) and different forms of NRG1; in all conditions, BrdU+ cells distribute in a typical pattern for an E24 MAM treated slice in that they are spread throughout cortical wall. n =  number of slices. Error bars  =  standard error. Two-way ANOVA followed by pairwise multiple comparison procedures (Holm-Sidak method). Scale Bar: 150 µm.
Mentions: To determine the ability of cells to migrate in organotypic cultures of either normal, E24 MAM treated cortex alone, or after coculture with HEK 293T cells embedded in Matrigel (as describe above), we plotted the distribution of BrdU+ cells after 2 DIC. Boundaries were drawn indicating the pia of a ferret slice and the outer edge of the VZ. This region was divided into 3 equal bins for each coculture and the number of cells per bin counted in a slab 500 µm in width in the somatosensory cortex. The bins correspond to the intermediate zone close to the VZ (i.e., the lowest part of the intermediate zone, IZL), a region in the IZ, but closer to the cortical plate (i.e., the upper part of the intermediate zone, IZU), and the region corresponding to the cortical plate (CP) (Figure 5c–d and Figure 7c–d). Histograms were made to indicate the position of BrdU+ cells across animals in each condition. To compare across samples, the number of cells/bin were calculated as a percent of the total number of cells in each slice.

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