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First published online 10 November 2004
doi: 10.1242/dev.01532

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3ß1 integrin modulates neuronal migration and placement during early stages of cerebral cortical development

¹ UNC Neuroscience Center and the Department of Cell and Molecular Physiology, The University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
² Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA
³ Department of Medicine, Children's Hospital, Boston and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA

View larger version (114K): [in a new window]   Fig. 1. Development of the preplate in 3 integrin-deficient mice. Neurons that are destined to the preplate of cerebral cortex were labeled at birth (E10.5 or E11.5) with BrdU, and their location was analyzed at E17, following the splitting of the preplate into marginal zone and subplate by invading migratory neurons. BrdU immunoreactivity was visualized with Cy3 and nuclei were counterstained with bis benzimide. When neurons were birthdated at E10.5, in wild-type mice, BrdU-labeled neurons were found in the marginal zone (arrows) and subplate layers (arrowheads) of cerebral cortex, as expected (A). Similar distribution was also seen in 3 integrin-deficient cortex (B). When neurons were birthdated at E11.5, most of the labeled neurons in wild-type cortex were found in subplate and few in the marginal zone (C). Identical pattern of labeling is also evident in 3 integrin-deficient cortex (D). Together, these results indicate that the preplate develops and splits normally in the absence of 3 integrin. MZ, marginal zone; CP, cortical plate; SP, subplate; IZ, intermediate zone. Scale bar: 60 µm.

View larger version (62K): [in a new window]   Fig. 2. Disrupted cortical neuronal placement in 3 integrin-deficient mice. 3 integrin-deficient mice were crossed with Thy1-GFP line-I (Feng et al., 2000), known to express GFP in layer VI neurons (A). At P0 (B), GFP expression is limited to distinct medial and lateral domains of the occipital region of the cortex. Expression of GFP was also noticed in the striatal area and in an area surrounding third ventricle (B). In wild-type lateral occipital cortex at P0 (C), layer VI neurons express GFP, whereas in 3 integrin-deficient cortex (D), GFP-positive neurons were ectopically placed, away from their destination in layer VI. Higher magnification images of GFP-labeled neurons in wild-type (C', arrowheads) and 3 integrin mutant (D', arrows) cortex show disrupted apical dendrite orientation in 3 integrin neurons. Immunolabeling with anti-calretinin or calbindin antibodies indicates malpositioning of cortical interneurons in 3 integrin mutants. In P0 wild-type cortex, anti-calretinin (E) or calbindin (G) antibodies primarily label bands of neurons in newly formed layers I-III and III/IV, respectively. By contrast, calretinin-(F) or calbindin (H)-expressing neurons are diffusely distributed in 3 integrin mutant cortex. OC, occipital cortex; ST, striatum; 3rd V, third ventricle; LV, lateral ventricle; CP, cortical plate; SP, subplate; IZ, intermediate zone; VZ, ventricular zone; WM, white matter. Scale bar: 100 µm in A; 75 µm in C,D; 60 µm in C',D'; 180 µm in E-H.

View larger version (67K): [in a new window]   Fig. 3. Disrupted neuronal migration in 3 integrin deficient cortex. Neurons in wild-type and mutant E15-E16 cortices were labeled with BAPTA green. Labeled cells in the intermediate zone of the slices, migrating in radial direction (A,B), towards the ventricular zone (C,D) or in tangential orientation (E,F) were repeatedly monitored. (A,C,E,G) In wild-type cortex, neurons migrated radially, tangentially and towards the ventricular zone at an average rate of 27±3.2 µm/hour, 43.5±5.4 µm/hour and 43±3.9 µm/hour, respectively. Arrowheads in A,C,E indicate sample migrating wild-type cells. (B,F,G) In 3ß1 integrin-deficient cortex, the rates of radial and tangential migration of neurons were significantly reduced to 16±1.6 µm/hour, 29±3.1 µm/hour, respectively. By contrast, no significant differences were noticed in the rate of ventricular zone directed neuronal migration (C,D; wild type, 43±3.9 µm/hour; mutant, 37±4.1 µm/hour). Arrows in B,D,F indicate sample migrating 3 integrin mutant cells. n=80 for radial wild type and mutant; n=80 for tangential wild type, n=75 for mutant; n=50 for wild type, ventricular zone directed, n=20 for mutant. Data shown are mean±s.e.m.; asterisk (G), significant when compared with controls at P<0.01 (Student's t-test). Time elapsed since the beginning of observations are indicated in minutes. P and V, direction of the pial and ventricular surfaces, respectively. Scale bar: 50 µm in A-D; 40 µm in E,F. (Also see the Figs S2, S3 and Movies 1, 2 in the supplementary material.)

View larger version (82K): [in a new window]   Fig. 4. Altered dynamics of leading and trailing edges of migrating neurons in 3 integrin-deficient cortex. Time-lapse imaging of GFP-labeled neurons in the wild-type cortex indicates active extensions and retractions of the leading (arrow) and trailing processes (arrowhead) of migrating neurons (A). By contrast, 3 integrin mutant neurons displayed reduced protrusive activity in their leading and trailing processes (B). Quantification of extensions and retractions (activity index) indicates a 28% reduction in 3 integrin mutant cells. Cells shown are from the intermediate zone of E16 cortex. Time elapsed since the beginning of observations are indicated in minutes. Number of cells analyzed: n=24, wild type; n=28, mutant. Data shown are mean±s.e.m.; asterisk (C), significant when compared with controls at P<0.01 (Student's t-test). Scale bar: 25 µm. (Also see Movies 3, 4 in the supplementary material.)

View larger version (56K): [in a new window]   Fig. 5. Deficient actin dynamics in 3 integrin mutant cortical cells. E14 cortical cells were electroporated with EGFP-actin and time-lapse images of the actin cytoskeleton at the leading edges was recorded after 48 hours. The actin cytoskeleton of the tips of the wild-type cells shows very active remodeling (A; see actin microspikes in regions marked with arrowheads, v indicates dynamic changes in their assembly and disassembly). (C) Higher magnification view of the outlined area in A. Arrows indicate actin microspikes undergoing remodeling. By contrast, actin cytoskeleton of leading edges of the 3-deficient cells display significantly reduced dynamic activity (B; compare actin microspikes in regions marked with arrowheads, compare v with similarly marked actin microspikes in A). (D) Higher magnification view of the outlined area in B. Arrows indicate actin microspikes that are considerably less dynamic than those from wild-type cells. (E) Re-expression of 3 integrin rescued actin dynamics deficits in 3 integrin mutant cells. Arrow indicates an actin microspike undergoing dynamic remodeling at the leading edge of an 3 mutant cell transfected with 3 integrin DNA. Time elapsed since the beginning of observations is indicated in minutes. Scale bar: 10 µm in A,B; 3 µm in C-E. (Also see Movies 5-7 in the supplementary material.)

View larger version (40K): [in a new window]   Fig. 6. Altered filopodial activity in the leading edges of 3 mutant cortical cells. E14 neuronal cells from wild-type or mutant cortices were electroporated with PH-Akt-EGFP (A,B) and time-lapse images of the growth cone ends of neuronal processes were recorded after 48 hours. PH-Akt-EGFP is targeted to active growth cones, thus enabling the evaluation of leading edge activity. In PH-Akt-EGFP transfected wild-type cells (A), filopodia emerge in large numbers from many spots along the leading edge (A; see activity in regions marked with asterisks) and appear to intensely sample the environment of the cell. By contrast, fewer filopodia emerge from the two adjacent leading edges (^, *) of PH-Akt-EGFP transfected 3 integrin mutant cortical cells (B), and their ability to probe the cellular environment appear to have been retarded (compare activity in regions marked with asterisks in A and B). Re-expression of 3 integrin in 3 integrin-deficient cells rescued the deficits in filopodial activity (C; see active region marked with an asterisk). n=67 (wild type), n=70 (mutant and mutant + 3 integrin). Time elapsed since the beginning of observations is indicated in minutes. Scale bar: 20 µm. (Also see Movies 8-10 in the supplementary material.)

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