Multiple roles of bone morphogenetic protein signaling in the regulation of cortical cell number and phenotype - PubMed (original) (raw)

Multiple roles of bone morphogenetic protein signaling in the regulation of cortical cell number and phenotype

P C Mabie et al. J Neurosci. 1999.

Abstract

Members of the bone morphogenetic protein (BMP) family have been implicated in multiple aspects of neural development in both the CNS and peripheral nervous system. BMP ligands and receptors, as well as the BMP antagonist noggin, are expressed in the developing cerebral cortex, making the BMPs likely candidates for regulating cortical development. To define the role of these factors in the developing cerebral cortex, we examined the effects of BMP2 and BMP4 on cortical cells in vitro. Cells were cultured from embryonic day 13 (E13) and E16 rat cerebral cortex in the absence or presence of different concentrations of fibroblast growth factor 2, a known regulator of cortical cell proliferation and differentiation. At E13, the BMPs promoted cell death and inhibited proliferation of cortical ventricular zone cells, resulting in the generation of fewer neurons and no glia. At E16, the effects of the BMPs were more complex. Concentrations of BMP2 in the range of 1-10 ng/ml promoted neuronal and astroglial differentiation and inhibited oligodendroglial differentiation, whereas 100 ng/ml BMP2 promoted cell death and inhibited proliferation. Addition of the BMP antagonist noggin promoted oligodendrogliogenesis in vitro, demonstrating that endogenous BMP signaling influences the differentiation of cortical cells in vitro. The distribution of BMP2 and noggin within the developing cortex suggests that local concentrations of ligands and antagonists define gradients of BMP signaling during corticogenesis. Together, these results support the hypothesis that the BMPs and their antagonist noggin co-regulate cortical cell fate and morphogenesis.

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Figures

Fig. 1.

Fig. 1.

Effects of BMP2 on the growth of cortical VZ cells plated at low density with FGF2 10 ng/ml. Photomicrographs of rat E13 cortical cells grown with FGF2 alone (A,D, G, J), FGF2 plus BMP2 10 ng/ml (B, E, H,K), or FGF2 plus BMP2 100 ng/ml (C, F, I,L). A–C, Phase-contrast images at 5 div.D–F, Bisbenzimide staining at 8 div.G–I, β tubulin III staining at 8 div.J–L, Nestin staining at 8 div. The same photographic field is shown for each condition in D,G, J; E, H,K; and F, I,L at 8 div. Arrows indicate examples of dying cells with apoptotic bodies (A–C), cells expressing β tubulin III-immunoreactivity (G–I), and nestin immunoreactivity (J–L). In each condition, an occasional cell coexpressed both β tubulin III and nestin (for example, the_top_ cell indicated with an arrow in_I_ and L). Scale bar: A–C, 8 μ

m

; D–L, 12 μ

m

.

Fig. 2.

Fig. 2.

Effects of BMP2 on the growth of cortical VZ cells. The number of total viable cells was quantitated at the indicated time points. A, Effects of BMP2 10–100 ng/ml on E13 cells grown at low density with FGF2 10 ng/ml for 8 div.B, Effects of BMP2 10 ng/ml on cells grown at moderate density with FGF2 0.1–10 ng/ml for 9 div. C, Effects of BMP2 0.1–100 ng/ml on E13 cortical cells grown at moderate density without FGF2 for 5 div. *p < 0.05; **p < 0.01.

Fig. 3.

Fig. 3.

Effects of BMP2 on E16 cortical cells grown at low density with FGF2 10 ng/ml. Photomicrographs of cells grown with FGF2 alone (A, C, E) or FGF2 plus BMP2 10 ng/ml (B, D,F) for 8 div. A, B, Bisbenzimide staining. C, D, β tubulin III staining. E, F, O4 staining. The same photographic field is shown for each condition. Scale bar, 12 μ

m

.

Fig. 4.

Fig. 4.

Effects of BMP2 on E16 cortical cells. The number of total viable cells and cells expressing neuronal (β tubulin III), oligodendroglial (O4), and neuroepithelial (nestin) markers was quantitated at the indicated time points. A, Effects of BMP2 on E16 cells grown at low density with FGF2 10 ng/ml.B, Effects of BMP2 on the differentiation of E16 cortical grown at low density with FGF2 10 ng/ml for 8 div.C, Effects of BMP2 0.1–100 ng/ml on E16 cortical cells grown at moderate density without FGF2. *p < 0.05; **p < 0.01.

Fig. 5.

Fig. 5.

Effects of delaying the addition of BMP2 on E16 cortical cell differentiation. Cells were grown at low density with FGF2 alone for the first 4 div, and then BMP2 was added at 10 or 100 ng/ml. The number of cells expressing neuronal (β tubulin III), oligodendroglial (O4), astrocytic (GFAP), and neuroepithelial (nestin) markers was quantitated at 8 div. *p < 0.05; **p < 0.01.

Fig. 6.

Fig. 6.

Effects of noggin on E16 cortical cells grown at low density with FGF2 10 ng/ml. Photomicrographs of cells grown with FGF2 alone (A, C) or FGF2 plus noggin 100 ng/ml (B, D) for 8 div. A,B, β tubulin III staining. C,D, O4 staining. Scale bar, 15 μ

m

.

Fig. 7.

Fig. 7.

Effects of noggin on E16 cortical cells grown at low density with FGF2 10 ng/ml. The number of cells expressing neuronal (β tubulin III), oligodendroglial (O4), and neuroepithelial (nestin) markers was quantitated at 8 div. *p < 0.05; **p < 0.01.

Fig. 8.

Fig. 8.

BMP2 and noggin expression in the developing cortex. Photomicrographs of E16 rat (A) and neonatal (B, C) murine cortex stained with a monoclonal antibody to BMP2 (A, B) and a monoclonal antibody to noggin (C). The arrows in A indicate the pial (top) and ventricular (bottom) surfaces. For neonatal coronal sections (B, C), the_arrows_ in C indicate the position of (from top to bottom) the pial surface, the border between cortical gray matter and developing subcortical white matter, the border between subcortical white matter and subventricular zone, and the lateral ventricle.

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