Beta1 integrins in radial glia but not in migrating neurons are essential for the formation of cell layers in the cerebral cortex - PubMed (original) (raw)

Beta1 integrins in radial glia but not in migrating neurons are essential for the formation of cell layers in the cerebral cortex

Richard Belvindrah et al. J Neurosci. 2007.

Abstract

Radial glial cells in the cerebral cortex serve as progenitors for neurons and glia and guide the migration of cortical neurons. The integrin alpha3beta1 is thought to mediate interactions of migrating neurons with radial glial cells and to function as a receptor for the reelin signaling molecule. Here, we challenge this view and demonstrate that beta1 integrins in migrating neurons are not essential for the formation of cell layers in the cerebral cortex. Cortical cell layers also form normally in mice deficient in the integrin alpha3beta1. However, we provide evidence that beta1 integrins in radial glia control the morphological differentiation of both glia and neurons. We conclude that beta1 integrins in radial glia are required for the proper development of the cerebral cortex, whereas beta1 integrins in migrating neurons are not essential for glial-guided migration and reelin signaling.

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Figures

Figure 1.

Analysis of Nex-CRE mice. Nex-CRE mice were crossed with Rosa26lacZ-loxP mice and analyzed for nuclear localized LacZ expression (blue). a, At E11, LacZ was expressed in cranial ganglia (arrow). b, c, At E12.5, LacZ was expressed in basal ganglia, the mid-hindbrain junction, and the telencephalon (arrows in b). Expression was also observed in the spinal cord (arrows in c). d, At E12.5 prominent LacZ expression was observed close to the ventricular zone (arrows) and in scattered cells throughout the cortical wall. e–i, Histological sections of adult mice. e, LacZ was prominently expressed throughout the telencephalon and in the olfactory bulb. f, In the olfactory bulb, LacZ was expressed in the mitral cell layer and in the periglomerular layer. g–i, LacZ was widely expressed throughout the cortical wall (g), in the hippocampus (h) and in a subset of cells in the internal granule cell layer in the cerebellum (i). Scale bars: a–c, 1 mm; d, 360 μm; e, 820 μm; f, 260 μm; g, 215 μm; h, 205 μm; i, 260 μm.

Figure 2.

Nex-CRE mice induce recombination in cortical neurons but not in glia. a, b, Sagittal sections through the cortex were stained for LacZ (blue) and GFAP (brown). GFAP-positive cells (arrowheads) were LacZ negative. Arrows point to LacZ-positive cells that were interspersed between GFAP-positive cells; cc, corpus callosum. d–f, Sagittal cortical sections were stained for LacZ (blue) and NeuN (brown). LacZ-positive cells were NeuN positive, confirming their identity as neurons. Note that LacZ staining in d–f was for a shorter time than in a and b. Therefore, the signal appeared less strong and less diffuse in d–f, revealing strong staining in one or two dots within nuclei (arrows in f). c, g, Cortical neurons and glial cells from P0 mice were cultured on PDL/LN substrates and stained. c, LacZ-positive cells (blue) were interspersed between GFAP-positive cells (brown). g, MAP2-positive neurons (red), expressed CRE (green) in nuclei. Scale bars: a, 330 μm; b, c, 23 μm; d, 115 μm; e, 30 μm; f, g, 18 μm.

Figure 3.

Characterization of NEX-CRE mice and analysis of Itgb1 expression by flow cytometry. a–n, Z/EG reporter mice carrying a CRE-inducible GFP transgene were crossed with NEX-CRE mice to analyze the CRE recombination pattern. a–c, GFP fluorescence was evident in the developing cerebral cortex of E12.5–E16.5 embryos by whole-mount analysis. d, GFP fluorescence throughout the cerebral cortex was also evident in vibratome sections. e–h, Coronal sections of mice at E14.5 and E16.5 were stained with antibodies to GFP. GFP expression was evident in the SVZ and cortical plate (CP), but not in the VZ. In e and g, nuclei were counterstained with DAPI (blue). i–k, Higher-magnification views of coronal sections stained with DAPI and antibodies to GFP. The vast majority of cells were GFP positive (arrows). l–n, Sections from E15.5 animals were stained with antibodies to doublecortin (dcx, red) and GFP (green). (l′–n′) Higher-magnification views of the area outlined in l–n. Note overlapping patterns of cytoplasmic staining for doublecortin and GFP. o, The expression levels of β1 integrins were evaluated in GFP-positive cells that had been isolated by FACS sorting from Nex-CRE mice containing a CRE-inducible GFP transgene (control), or from Itgb1-NEXko mice carrying a CRE-inducible transgene (mutant). The green line shows samples incubated with antibody to β1 integrins (β1 Ab); in the blue line samples, the secondary antibody alone was added (second); and in the red line samples, no antibody was added. Scale bar: a, 600 μm; b, c, 3.75 mm; d, 2 mm; e, f, 150 μm; g, h, 300 μm; i–k, 37.5 μm; l–n, 150 μm; l′–n′, 37.5 μm.

Figure 4.

Analysis of the cerebral cortex of adult Itgb1-NEXko mice. a, b, Sagittal section through the cerebral cortex of Itgb1-NEXko mice and control littermates (wt, wild-type) at P60 were stained with Nissl. The overall morphology of the cerebral cortex appeared unaffected in the mutant mice. c, Cell density was determined in layer II/III in three wild-type and three Itgb1-CNSko mice. There was no difference in cell density. d, e, The thickness of the cerebral cortex was determined at five anatomical levels as indicated in the diagram. There was no significant difference between wild-type and Itgb1-CNSko mice. Scale bars: a, b, 400 μm.

Figure 5.

Cortical layers form normally in Itgb1-NEXko mice. a–f, Sagittal sections through the cerebral cortex of wild-type, Itgb1-CNSko, and Itgb1-NEXko mice at P60 were stained with antibodies to CUX1 (brown), to reveal layer II–IV neurons. CUX1-positive neurons in Itgb1-NEXko mice were confined to cortical layers II–IV. CUX1-positive neurons in Itgb1-CNSko mice were also present in cortical layers II–IV, but the layer had a wavy appearance and appeared to be broader than in wild-type mice. g–m, Sagittal sections through the cerebral cortex were stained with an antibody to a nonphosphorylated form of neurofilament (Smi32) (brown), which specifically stains a subset of layer III and V neurons, as well as a subset of cells in the hippocampus. Neurons in wild-type, Itgb1-CNSko mice, and Itgb1-NEXko mice were appropriately located in layer III and V, but layer in Itgb1-CNSko mice appeared wavy (g–i). Higher-magnification views revealed that dendritic process in layer III (j, k) and the hippocampus (l, m) were not noticeably affected in Itgb1-NEXko mice. Scale bars: a–c, 400 μm; d–f, 125 μm; g–i, 450 μm; j–m, 70 μm.

Figure 6.

Reactive gliosis in Itgb1-CNSko mice. a–c, Histological sections were stained at P60 with antibodies to GFAP and processed with secondary antibodies coupled to peroxidase. In Itgb1-CNSko mice, astrocytes that strongly express GFAP (brown) were abundantly present. d–k, GFAP expression was evaluated by immunofluorescence microscopy (green) at different postnatal ages. At P0, GFAP-positive cells were detectable in the meninges and corpus callosum close to the VZ of wild-type (d) and Itgb1-CNSko mice (g). At P10, patches of astrocytes expressing high amounts of GFAP started to be detectable within the cortical wall of Itgb1-CNS knock-out mice (h), but not in wild-type mice (e). GFAP-positive cells were much more abundant in Itgb1-CNSko mice by 1 year of age and distributed throughout the cerebral cortex (f, i). j, k, High-magnification view of the cortex of mice at 1 year of age. Note that the GFAP-positive cells in Itgb1-CNSko mice show the characteristic morphology of astrocytes. Scale bars: a–c, 60 μm; d–i, 200 μm; j, k, 50 μm.

Figure 7.

Histological analysis of the cerebral cortex at P0 in Itga3_-null mice. a–d, Overall brain morphology and the organization of the cerebral cortex appeared unaltered when analyzed by Nissl staining. e, f, Cell density in layers V/VI and cortical thickness (at similar anatomical levels as shown in Fig. 4_d) was not significantly altered in _Itga3_-null mice. Scale bars: a, b, 425 μm; c, d, 690 μm.

Figure 8.

The cortical marginal zone is unaffected in _Itga3_-null mice. Sagittal sections through the cerebral cortex of wild-type and _Itga3_-null mice at P60 were stained with antibodies to βΙΙΙ-tubulin (Tuj1 clone, green), to reveal overall organization of the cortex (a, b); calretinin (calret, red), to visualize cells in the cortical marginal zone and interneurons that migrate into cortical layers (arrows in e, f) (c–f); reelin (green), to reveal Cajal–Retzius cells (nuclei are stained with DAPI and shown in blue) (g, h). Scale bars: a, b, 70 μm; c, d, g, h, 80 μm; e, f, 40 μm. mz, Marginal zone.

Figure 9.

Cortical layers form normally in _Itga3_-null mice. Sagittal sections through the cerebral cortex of wild-type and _Itga3_-null mice at P0 were stained with antibodies to TBR1, to reveal neurons in layers II–IV and VI (a–d); and CUX1, to reveal neurons in layers II–IV (e, f). Note that there was no difference in the distribution of different neuronal subtypes in wild-type and _Itga3_-null mice. Scale bars: a, b, 425 μm; c, d, 80 μm; e, f, 100 μm.

Figure 10.

Defects in neuronal and glial process outgrowth. a–c, Sections through the cerebral cortex of P0 animals of the indicated genotype were stained with antibodies to MAP2 (green), to reveal dendrites. Note that dendrite morphology was severely affected in Itgb1-CNSko mice only. d–l, Dissociated cells from the cerebral cortex of P0 animals of the indicated genotype were plated onto PDL/LN substrates and cultured for 1 d (1div) or 5 d (5div). The cultures were stained with antibodies to MAP2 or GFAP (brown), as indicated. d–f, In cultures containing cells from wild-type mice, neurite outgrowth was evident at 1div and more pronounced by 5div. Many glial cells also formed extensive projections (arrows in f). g–i, Neurite outgrowth and the formation of glial processes was severely impaired in primary cultures containing cells from Itgb1-CNSko mice. j–l, Neurite outgrowth and the formation of glial processes were restored in cultures with cells from Itgb1-NEXko mice. m, n, The length of the glial processes and neurites was determined. Values indicated the mean and SD. A Student's t test was performed (***p < 0.01). Scale bars: a–c, 50 μm; d–l, 76 μm.

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Beta1 integrins in radial glia but not in migrating neurons are essential for the formation of cell layers in the cerebral cortex - PubMed (original) (raw)