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First published online July 27, 2006
doi: 10.1242/10.1242/dev.02486

Development 133, 3245-3254 (2006)
Published by The Company of Biologists 2006
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Basement membrane attachment is dispensable for radial glial cell fate and for proliferation, but affects positioning of neuronal subtypes

Nicole Haubst1, Elisabeth Georges-Labouesse2, Adele De Arcangelis2, Ulrike Mayer3 and Magdalena Götz1,4,*

1 Institute for Stem Cell Research, GSF, National Research Center for Environment and Health, Ingolstädter Landstr.1, D-85764 Neuherberg/Munich, Germany.
2 IGBMC, CNRS/INSERM/ULP, BP 163, 67404 Illkirch, CU de Strasbourg, France.
3 Biomedical Research Centre, School of Biological Sciences, University of East Anglia, Norwich, UK.
4 Department of Physiology, Ludwig-Maximilians University, Munich, Schillerstr. 46, D-80336, Munich, Germany.


Figure 1
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  		Fig. 1. Basement membrane disruption and radial glial endfeet detachment in laminin {gamma}1III4-/-, perlecan-/- and {alpha}6-integrin-/- cortex at midneurogenesis. Fluorescent micrographs of immunostained (as indicated in the panels) frontal sections of telencephali of embryonic day (E) 14 wild-type and mutant littermates. (A-D) Laminin immunoreactivity underneath the pial surface (arrowheads in A) and surrounding the blood vessels (arrows in A). Insets (a-d) depict representative high-power views of the BM in the respective mouse line. Arrowheads in C,D indicate disruptions of the BM. (E-J) The absence of BLBP-immunoreactive radial glia endfeet (arrowhead in E,G) in the laminin {gamma}1III4-/- (F,H), perlecan-/- cortex (I) and {alpha}6 integrin-/- cortex (CTX; J) compared with wild type (E,G). Ectopic BLBP-positive cell somata in the laminin {gamma}1III4-/- cortical plate (CP) (arrows in F,H). The broken white line (E) indicates the ventricular surface (VS). CTX, cerebral cortex; GE, ganglionic eminence; VZ, ventricular zones. Scale bars: 200 µm in A-D; 50 µm in E,F; 25 µm in G,H; 100 µm in I,J.

 

Figure 2
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  		Fig. 2. Proliferation, interkinetic nuclear migration and orientation of cell division in wild-type and laminin {gamma}1III4-/- cortex at midneurogenesis. Fluorescent micrographs of E14 wild-type and laminin {gamma}1III4-/- cortex immunostained as indicated (A,B,E-H) reveal similar numbers of precursors (Ki67-positive) in mitosis (PH3-positive) at the ventricular surface (VS, arrowheads in A) or at abventricular positions. Ectopic clusters of precursor cells within the laminin {gamma}1III4-/- cortical plate (CP) are indicated by arrows in B. (C) Histogram depicting the percentages of precursors dividing at the ventricular surface (VZ in C), subventricular zone (SVZ) or at ectopic positions assessed by the quantification of PH3-positive cells at E14 (wild type: n=40 sections, two animals, laminin {gamma}1III4-/-: n=45, two animals). (D) Histogram depicting the number of PH3-positive cells per cortex area (100 µm2) dividing at VZ or SVZ positions respectively (wild type: n=43, two animals; laminin {gamma}1III4-/-: n=51, two animals). (E-H) BrdU-immunostaining (red) reveals cells in S phase (0.5 hours after BrdU-injection; E,F) and 6 hours after S-phase labelling when they have moved towards the ventricular surface (G,H). (I) A dividing cell in anaphase labelled with propidium iodide. The angle of cell division was assessed by measuring the angle between a line at the VS and the separating chromatids. (J) Histogram depicting the percentages of cells dividing horizontally with respect to the VS (0-30°), obliquely (30-60°) and perpendicularly (60-90°) in E14 cortex (wild type: n=103 mitoses, two animals; laminin {gamma}1III4-/-: n=110, two animals). Scale bars: 100 µm

 

Figure 3
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  		Fig. 3. Neurogenesis in wild-type and laminin {gamma}1III4-/- cortex. (A-H) E14 or E18 wild-type and laminin {gamma}1III4-/- cortex sections stained for the neuronal antigen ßIII-Tubulin or Math2 mRNA. Arrowheads in B and red arrows in H indicate neuronal ectopias in the subarachnoidal space, arrows in B indicate ectopic clusters of precursors in the laminin {gamma}1III4-/- cortex. The broken yellow and red lines in C delineate the cortical plate thickness in relation to the cortex thickness. Math2 expression is absent in the upper layers of the laminin {gamma}1III4-/- cortex (H), while these neurons still contain ßIII-Tubulin (F). Scale bars: 100 µm for A,B,E,F; 200 µm for C,D,G,H.

 

Figure 4
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  		Fig. 4. Neuronal subtypes in the E18 laminin {gamma}1III4-/- cortex. Micrographs of in situ hybridization for the layer-specific mRNAs of Cux2, Rorb (RORß) and Er81 (A-F), and immunostaining for interneurons (G-J) in E18 wild-type and laminin {gamma}1III4-/- cortex. Cux2- and Rorb-expressing layer II-IV neurons are misplaced to the position of Er81-positive deep layer neurons in the laminin {gamma}1III4-/- cortex (E,F). The red arrows indicate the layer specific gene expression. Conversely, GABAergic interneurons containing calretinin (red in G,H) or calbindin (red in I,J) were detected in the outer part of the laminin {gamma}1III4-/- cortex (H,J) in contrast to their scattered position in the wild-type cortex (G,I). Early born reelin-immunoreactive cells (green in G-J) are mostly calretinin positive (G',H') and are not increased in the laminin {gamma}1III4-/- cortex (H) compared with wild type (G). Scale bars: 200 µm in A-F; 50 µm in G-J.

 

Figure 5
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  		Fig. 5. Proliferation and neurogenesis in {alpha}6 integrin-/- cortex. Micrographs of E14 wild-type and {alpha}6 integrin-/- cortex sections (A,B,E-H) stained as indicated. (C,D) Quantification of the number of PH3-positive cells in M phase per cortex area (C; wild type: n=39 sections, three animals, {alpha}6 integrin-/-: n=50, three animals) and of the percentages of cells dividing with horizontal, oblique or perpendicular orientation to the ventricular surface (D; wild type: n=89 mitoses, one animal, {alpha}6 integrin-/-: n=150, one animal) in wild-type and {alpha}6 integrin-/- cerebral cortex. Scale bars: 100 µm.

 

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© The Company of Biologists Ltd 2006