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3ß1 integrin modulates neuronal migration and placement during early stages of cerebral cortical developmentFiles in this Data Supplement:
Fig. S1. Neuronal displacement in a3 integrin-deficient mice. To quantify GFP- (line I), calretinin- or calbindin-positive neuronal distribution, cerebral cortex was divided into ten equal bins from the ventricular surface (VS) to the pial surface (PS), and the percentage of labeled cells in each area was measured. This indicates the ectopic placement of neurons in a3 integrin-deficient cortex. Data shown are mean±s.e.m. Labeled cells were counted in ten sections of the occipital region of the cerebral cortex from five embryos each of wild type and a3 integrin mutant mice.
Fig. S2. Assays for neuronal migration. Neurons in wild-type and mutant E15-E16 cortices were labeled with BAPTA green. Incubation of the cortical slices with BAPTA green labels cells in the entire cerebral wall, including neurons migrating in different orientations (A). By contrast, application of BAPTA green to the ganglionic eminence (GE) specifically labels neurons migrating tangentially into the cerebral wall (arrow, B). CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone. Scale bar: 50 mm in A; 27 mm in B.
Fig. S3. Migrating neurons in wild type and a3 integrin-deficient cerebral cortex. Migration of BAPTA green labeled neurons in wild type (A) and a3 integrin-deficient (B) embryonic cortex (intermediate zone region) was repeatedly imaged at 5-10 minute intervals using a Zeiss inverted microscope attached to a PASCAL confocal laser scanning system and a live cell incubation chamber. These images were compiled as AVI movie files to illustrate the differences in migration between wild-type and a3 integrin-deficient neurons. Sample cells migrating in distinct orientations are indicated. In wild-type cortex, neurons migrated radially, tangentially and towards ventricular zone at an average rate of 27±3.2 mm/hour, 43.5±5.4 mm/hour and 43±3.9 mm/hour, respectively. In a3b1 integrin-deficient cortex, the rates of radial and tangential migration of neurons were significantly reduced to 16±1.6 mm/hour, 29±3.1 mm/hour, respectively. In contrast no significant differences were noticed in the rate of ventricular zone directed neuronal migration (wild type=43±3.9 mm/hour, mutant=37±4.1 mm/hour).
Fig. S4. Enhanced adhesion of a3 integrin-deficient cortical neurons to fibronectin substrate. E16 wild-type (A,C) and a3 integrin–/– (B,D) cortical cells were plated on laminin (A,B) or fibronectin (C,D) coated 24-well plates (50,000 cells/well plating density). After an hour in vitro, adherent neurons were labeled with neuron specific Tuj-1 antibodies (red, A-D) and quantified. A 62% increase in the adhesion of a3 integrin-deficient neurons was noticed on fibronectin substrate (C-E), whereas no significant difference in adhesion was evident on laminin substrate (A,B,E). Asterisk (E), significant when compared with controls at P<0.01 (Student’s t-test). Data shown are mean±s.e.m., based on four independent experiments. Scale bar: 85 mm.
Movie 1. Wild-type neuronal migration (see Fig. 3).
Movie 2. Migration of a3 integrin mutant neurons (see Fig. 3).
Movie 3. Wild-type neuron. Leading and trailing edge activity (see Fig. 4).
Movie 4. a3 integrin mutant neuron. Leading and trailing edge activity (see Fig. 4).
Movie 5. Wild-type actin dynamics at the leading edge (see Fig. 4).
Movie 6. a3 integrin mutant actin dynamics at the leading edge (see Fig. 5).
Movie 7. Actin dynamics at the leading edge of a3 integrin mutant cell transfected with a3 integrin (i.e. a rescued cell) (see Fig. 5).
Movie 8. Wild type PH-Akt-EGFP (see Fig. 6).
Movie 9. a3 integrin mutant PH-Akt-EGFP (see Fig. 6).
Movie 10. Ph-Akt-EGFP at the leading edge of a3 integrin mutant cell transfected with a3 integrin (i.e. a rescued cell) (see Fig. 6).
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