Figure 1. Normal tissue development and prolonged bleeding in Fn1^syn/syn mice.

(A) Cartoon of FN and the nucleotide point mutations disrupting the function of the synergy site. (B) Representative images of 3-months-old Fn1 ^+/+ and Fn1^syn/syn heart sections stained with H and E and immunostained for FN. (C) Confocal images of ear whole-mounts from 3 months-old mice immunostained with anti-PECAM-1 and anti-αSMA to visualize the dermal endothelial cell tubes and smooth muscle cells. (D) Bleeding time of 3-months-old Fn1^+/+ (n = 11) and Fn1^syn/syn (n = 11) mice. (E) Platelet counts in blood samples of Fn1^+/+ (n = 18) and Fn1^syn/syn (n = 19) mice. (F) FN content in platelets derived from Fn1^+/+ (n = 6) and Fn1^syn/syn (n = 6) mice relative to their vinculin levels. (G) Occlusion time of injured arterioles in the cremaster muscle of 3-months-old Fn1^+/+ (n = 11) and Fn1^syn/syn (n = 11) mice. (H) Representative still images of the arteriolar occlusion (white:platelets). Values are shown as mean ± SD; statistical significances were calculated using the Student t-test; **p<0.01 and ***p<0.001.

DOI: http://dx.doi.org/10.7554/eLife.22264.002

Figure 1—figure supplement 1. Strategy used to generate the Fn1^syn/syn mice and tissue and platelet analysis.

(A) Scheme of the FN gene and the targeting vector to generate the mouse with a dysfunctional synergy site. The synergy region is located in the FNIII9 and encoded in exon 28, shown in red. (B) The homologous recombination of the targeting vector was re-tested in ES cell clones 56 and 266 by Southern-blot using probe 1 SacI digested DNA. (C) Mice were genotyped by PCR using primers shown as arrows in (a). (D) Liver, kidney and lung sections from 3-months-old Fn1^+/+ and Fn1^syn/syn mice stained with H and E (scale bar, 100 μm) and immunostained for FN (scale bar, 50 μm). (E) Ear whole-mount staining of laminin (Lam), FN, collagen IV, and erythrocytes (Ter119) to analyze the sub-endothelial ECM composition and the integrity of blood vessels (scale bars, 50 μm and 25 μm for FN immunostaining).

DOI: http://dx.doi.org/10.7554/eLife.22264.003

Figure 1—figure supplement 2. FN levels in platelets and blood from Fn1^syn/synmice and platelet aggregation assays.

(A) Western-blot to estimate FN levels in non-activated, washed platelets from Fn1^+/+ (n = 6) and Fn1^syn/syn mice (n = 6). (B) Western-blot to calculate FN and fibrinogen (Fg) levels in blood plasma from Fn1^+/+ (n = 6) and Fn1^syn/syn mice (n = 6). The first two lanes are commercial Fg and FN, respectively. (C–E) Representative in vitro aggregation assays using washed platelets from Fn1^+/+ (n = 6) and FN^syn/syn mice (n = 8). Aggregation curves of platelets activated with 5 μg/ml collagen (C), with 0.5 u/ml thrombin (D) or with 20 μM ADP (E).

DOI: http://dx.doi.org/10.7554/eLife.22264.004