The Journal of Neuroscience, April 25, 2007, ():
IQGAP1 Regulates Adult Neural Progenitors In Vivo and Vascular Endothelial Growth Factor-Triggered Neural Progenitor Migration In Vitro
J. Neurosci. Balenci et al.
27: 4716
Supplemental Data
Files in this Data Supplement:
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Supplemental Figure 1. In vitro comparison of intrinsic migration and differentiation capacities between wild-type and Iqgap1-null neurosphere cells.
Wild-type and Iqgap1-null neurospheres were plated on poly-L-lysine-coated glass slides in neurosphere basal medium supplemented with 3% fetal bovine serum (FBS).
A: Phase contrast images of time lapse imaging (0-2h) of wild-type and Iqgap1-null mutant neurospheres that attached to the substratum.
B-C: After 2, 4, 7 and 15 days in culture (DIV) cells were triple immunostained with neural cell markers GFAP, O4 and Tuj-1 (Gritti et al. 2002; Shen et al. 2004) and analyzed by confocal microscopy. (B), representative triple immunostaining of Iqgap1-null neurosphere after 15 DIV. (C), quantitative analysis of the different cell populations in wild-type and Iqgap1-null cultures.
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Supplemental Figure 2:
A-B:CLIP-170 is moderately expressed in mouse neurospheres. A: Comparison of CLIP-170 expression level in 10 ?g of total protein extracts from mouse 3T3 cells (lane 1), wild-type mouse neurospheres (lane 2), and Iqgap1-null neurospheres (lane 3). Proteins were resolved on 6%SDS-PAGE, and analyzed by Western Blot using anti-CLIP-170, anti-IQGAP1 and anti-?-catenin antibodies as indicated.
B: Characterization of IQGAP1 immunoprecipitates from neurospheres stimulated with VEGF. Total neurosphere extracts from wild-type (lane 1) and Iqgap1-null neurosphers (lane 6) and IQGAP1 immunoprecipitates from wild-type neurospheres not stimulated (lane 2) or stimulated with VEGF for 10 min (lane 3) or 30 min (lane 4) and from Iqgap1-null neurospheres stimulated with VEGF for 10 min (lane 5) were analyzed by Western blot with anti-IQGAP1, anti-CLIP-170 and anti-Lis1 antibodies as indicated.
C: IQGAP1 is not phosphorylated in VEGF-stimulated neurospheres. Total neurosphere extract (lanes 1) and IQGAP1 immunoprecipitates from wild-type neurospheres not stimulated (lanes 2) or stimulated with VEGF for 10 min (lanes 3) or 30 min (lanes 4) were analyzed by Western blot with: anti-IQGAP1 (a), anti-Phospho Ser-Thr (b) and anti-Phospho Tyr antibodies (c). The phosphoprotein band in total neurosphere extract (lane 1) in panel c, migrating in close proximity to IQGAP1 most likely correspond to auto-phosphorylated EGF receptor.
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Supplemental Figure 3. VEGF stimulation promotes transient Ca2+ increase in wild-type neurosphere cells.
A: VEGF-dependent Ca2+- signals were evaluated using the Ca2+-sensitive fluorescent dye Fluo-4. Neurospheres were plated onto poly-L-lysine substratum for 45 min and then loaded with 10?? Fluo-4- AM (Molecular Probes) in DMEM/F12 medium (Gibco) at 37°C for 20 min. After two extensive washes neurospheres were kept for at least 20 min in DMEM/F12 at 37°C for a complete de-esterification of the dye. Then, neurospheres were bathed in Tyrode saline solution containing: 136 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, pH 7,4 (NaOH) during the experiment at room temperature. Culture dishes were mounted on the stage of an upright Olympus BX51WI microscope equipped with a water immersion 20x objective lens (0.95 NA). The excitation light was provided by a 100 W mercury lamp. Fluorescent images were captured by a cooled digital CCD MicroMax Princetown camera (782 x 582 pixels). Images were acquired every 5 s with the software MetaFluor (v4.5, Universal Imaging). The shutter was controlled by the shutter driver Uniblitz VMM-D1 (Vincent Associates). The excitation light for Fluo-4 was filtered through a 460-495 nm excitation filter and the emitted light was collected through a 510-550 nm filter. After 1 min neurospheres were stimulated or not with VEGF (20 ng/ml). The figure shows a representative Fluo-4 signal recorded before and during the application of VEGF. It produced a long-lasting but transient elevation of the cytoplasmic concentration of Ca2+ in all the cells tested (n = 30). On average, VEGF (20 ng/ml) increased the Fluo-4 fluorescence by 55 +/- 7.5 % (mean +/- sem).
B: MAPTA action on wild-type neurospshere response to VEGF stimulation. Neurospheres were pre-incubated in the basal neurosphere culture medium in the presence of the cell-permeable calcium chelator MAPTA-AM (50?M) for 1h at 37°C. Then, the culture medium was removed and neurospheres were washed twice with fresh medium and were stimulated with VEGF (20 ng/ml). Phase contrast images at t=0 and t=6h shows that calcium chelation inhibits migratory response of wild-type neurospshere cells upon VEGF stimulation.
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Supplemental Figure 4. Iqgap1-null mice show no obvious defects in neuroblast migration.
Two wild-type (A) and Iqgap1-null (B) mice from the same litter received three succesive BrdU injections (1 per hour). Animals were sacrificed 1h after the last injection (t: 0), or six days (t: 6d) or 12 days (t: 12d) after injection. Coronal sections (20 ?m of thickness) of olfactory bulbs were stained with Hoechst for DNA (panels a, c, e) and double immunostained with anti-BrdU (green) and anti-PSA-NCAM (red) antibodies (panels b, d, f). One hour after the last injection (panels a-b), only few BrdU+/PSA-NCAM+ cells were found in the distal RMS. Some BrdU+/PSA-NCAM- cells (arrowhead) are juxtaposed to the chains of migrating neuroblasts (PSA-NCAM+) and correspond to neural progenitors (Gritti et al., 2002). After a 6 day-chase (panels c-d), BrdU+ cells within the distal RMS drastically increased corresponding to neuronal precursors/neuroblasts migrating to the OB. Some BrdU+/PSA-NCAM+ neuroblasts already reached the OB granular layers (arrowhead). Comparison between wild-type and Iqgap1-null mice revaled no significant difference in the timing of migration of BrdU-labeled neuroblasts to distal RMS. Atfter 12 day-chase (panels e-f), most of the BrdU-labeled neuroblasts have migrated to the granular and interneuron layers where they differentiated into mature neurons (BrdU+/PSANCAM-). Comparison between wild-type and Iqgap1-null mice revealed no difference in the timing of migration of BrdU-labeled neuroblasts to the OB granular layers.
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Video-1: Time lapse imaging of wild-type neurospheres stimulated with VEGF (20 ng/ml).
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Video-2: Time lapse imaging of Iqgap1 null neurospheres stimulated with VEGF (20 ng/ml).
