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First published online 31 May 2005
doi: 10.1242/jcs.02396

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Regulation of neural progenitor proliferation and survival by ß1 integrins

Dino P. Leone^1,*,, João B. Relvas^1,*, Lia S. Campos^2,, Silvio Hemmi³, Cord Brakebusch⁴, Reinhard Fässler⁴, Charles ffrench-Constant² and Ueli Suter^1,¶

¹ Institute of Cell Biology, Department of Biology, Swiss Federal Institute of Technology, ETH Hönggerberg, CH-8093 Zürich, Switzerland
² Departments of Medical Genetics and Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK
³ Institute of Molecular Biology Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland
⁴ Department of Molecular Medicine, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany

View larger version (48K): [in a new window]   Fig. 1. Generation of ß1-integrin null neurosphere cultures from the forebrain of neonatal mice. (A) Schematic representation of the conditional ß1-integrin allele (lox) and Cre recombinase driven recombination leading to the recombined null () allele. Exons are indicated by purple boxes, loxP sites by black triangles. Note the promoterless lacZ gene trailing the conditional allele with a splice acceptor site (SA) derived from the intron upstream of exon 2 preceding it. This leads to the expression of ß-gal in recombined cells under the control of the endogenous ß1-integrin promoter. (B) Schematic drawing of the experimental set-up used in this work. Primary neurospheres (2a) were obtained from neonatal forebrains of conditional ß1-integrin null mice. Neurospheres were trypsinized and the single cell suspension exposed to an adenovirus expressing the Cre recombinase (Ad-Cre; panel 2b). After an additional 10 days, primary infected neurospheres were harvested (3a) and either stained for X-gal or passaged (3b) to obtain further generations of infected neurospheres (4). (C) After infection with adeno Cre virus, the expression of ß-gal, monitored by X-gal staining, indicates the recombination of the ß1-integrin flox allele. Note that over 95% of the neurospheres are ß-gal-positive. (D) 12 µm cryostat section of control wild-type neurospheres stained for ß1-integrin. (E-G) Confocal microscopy analysis of 12 µm cryostat sections of recombined lox/wt spheres shows colocalization of ß-gal (reporting ß1-integrin promoter activity; green in E) and nestin (red in F) as yellow in G. Note that ß1-integrin staining shown in D is topographically similar to that of ß-gal in recombined lox/wt neurospheres. Bars, 150 µm (C); 50 µm (D-G).

View larger version (25K): [in a new window]   Fig. 2. Loss of ß1-integrin surface expression of neurosphere-derived cells, confirmed by FACS analysis using a FITC-conjugated anti-ß1-integrin antibody, reduces adhesion to fibronectin and laminin substrates. (A) Histogram of unlabelled wild-type neurospheres used as controls. (B) Histogram of non-infected, non-recombined neurospheres carrying the intact conditional ß1-integrin allele showing ß1-integrin surface expression (arrow). (C) Histogram of adeno Cre infected, recombined neurospheres demonstrating that the removal of the conditional ß1-integrin allele leads to cell surface loss of ß1-integrin expression (arrow). Two passages were needed for this dramatic reduction of ß1-integrin expression (D-E). Absence of ß1-integrin cell surface expression leads to reduced ß1 integrin-mediated short term adhesion of mutant cells (triangles) compared with control cells (diamonds) on both laminin-1 (LM1, D) and fibronectin (FN, E) substrates. (D) A significantly reduced adhesion (***P<0.001) was found on laminin-1 at all time points investigated compared with adherence of control cells. (E) On fibronectin a significant reduction in adhesion (***P<0.001) was found at 30 and 60 minutes after plating compared with that in control cells at the same time points. The y-axis represents the percentage of adherent cells compared with adherent cells on PDL-coated sister plates at the specific time points. Results are the mean±s.d. of three experiments.

View larger version (14K): [in a new window]   Fig. 3. Loss of ß1 integrin does not impair neural stem cell self-renewal as assessed by neurosphere formation over several passages. (A) No difference was found in the percentage of X-gal-positive recombined neurospheres between mutants and controls at passages (P) 1, P2 and P4 in the presence of 10 ng/ml of EGF and 20 ng/ml of FGF-2. The absence of a significant genotype-specific reduction of recombined neurospheres over several passages suggests that ß1 integrins are not essential for maintenance of neural stem cells. (B) The percentage of X-gal-positive neurospheres also did not significantly vary when the spherogenic assay was carried out at 1, 5 and 10 ng/ml EGF. This confirms that the loss of ß1-integrin expression does not affect the capacity of neurosphere formation. Note that 0.1 ng/ml EGF was too low for both mutant and control cells to support the formation of neurospheres. Results are the mean±s.d. of four experiments in A and three experiments in B.

View larger version (28K): [in a new window]   Fig. 4. A significant decrease in the diameter of mutant neurospheres is caused by a concomitant increase in cell death and a decrease in cell proliferation of nestin-positive progenitors. (A) The number of both mutant and control neurospheres was determined for each individual diameter ranging from 10 to 90 µm. The percentage of small (10-30 µm) neurospheres present in the mutant populations is significantly elevated in relation to control populations. In parallel, the mean percentage (±s.d.) of larger spheres (40-70 µm) is significantly reduced in ß1 integrin-deficient neurospheres compared with the value in controls (n=4; *P<0.05; **P<0.01; ANOVA). (B) The cellular composition of mutant neurospheres is different from control populations. In mutant neurospheres, the percentages of both GFAP-positive astrocytes and ß III-tubulin-positive neurons are increased whereas the number of nestin-positive progenitors is reduced. (C) The percentage of proliferating cells, stained with an antibody against phosphorylated histone H3, is significantly decreased in mutant neurospheres. (D) The number of apoptotic, TUNEL-positive cells is significantly increased in mutant neurospheres. (E) The percentage of apoptotic nestin-positive progenitors is significantly increased in the mutant neurospheres. Mean percentages±s.d. are shown for three separate experiments (D-E); *P<0.05, ***P<0.001 compared with respective values in control neurospheres (B-E).

View larger version (50K): [in a new window]   Fig. 5. The capacity to maintain nestin-positive progenitors within individual neurospheres is modulated by ß1-integrin expression. (A) The presence of at least one nestin-positive cell was assessed for each plated ß1-deficient neurosphere after 6 days of differentiation in the absence or in the presence of growth factors. In the absence of exogenous growth factors, the number of clones containing at least one nestin-positive cell is significantly decreased (***P<0.001) compared with control clones on laminin-1 (LM1), fibronectin (FN) or PDL substrates. By contrast, 85-95% of the mutant clones cultured in limiting growth factor conditions (3.3 ng/ml EGF, 2.0 ng/ml FGF, 10 ng/ml NGF) contained at least one nestin-positive progenitor cell. Results are the means±s.d. of three separate experiments. (B-C) Representative pictures of clones derived from control neurospheres (B) and from mutant neurospheres (C), cultured in the absence of growth factors for 6 days, and stained for nestin (red) and DAPI (blue). Bar, 70 µm.

View larger version (51K): [in a new window]   Fig. 6. ß1 integrin-deficient cells show impaired migration on both fibronectin and laminin-1. Intact neurospheres were plated and cultured in the absence or presence of exogenous growth factors on either fibronectin (FN), laminin-1 (LM1) or PDL. After 5 days, cell migration from individual mutant and control neurospheres was measured as described in Materials and Methods. (A) A significant decrease in the migration capacity of mutant cells is found on both fibronectin and laminin-1, whereas no difference is detected on PDL. Addition of growth factors (3.3 ng/ml EGF, 2.0 ng/ml FGF, 10 ng/ml NGF) partially restores the migration capacity of neurosphere mutant cells on laminin, but not on fibronectin. Note that the increase in the number of migrating cells in the presence of added growth factors does not reflect an increase in the cell migration index (see Materials and Methods). Results are the mean migration index±s.d. of three separate experiments; **P<0.01, ***P<0.001 when compared with the index in the relevant control cells. (B-M) Representative images of cells migrating from neurospheres cultured in the absence of growth factors (B-G) and in the presence of growth factors (H-M). Note the striking absence of migrating cells in the mutants plated on fibronectin (E) and the increase in migration in the mutants cultured with growth factors (L) on laminin-1 compared with the same genotype in the absence of growth factors (F). Bar, 200 µm.