Fig. 1. FN matrix organization is altered by FAK-deficiency. (A) FAK^-/- and control FAK^+/+ mouse endothelial cells were isolated from embryos and embryoid bodies. Endothelial cells isolated from mouse embryos were differentiated in vivo and were immortalized by p53-deficiency. Cells isolated from embryoid bodies were differentiated in vitro and were immortalized with polyoma middle T. FN staining in permeabilized FAK^-/- cells has a granular appearance and an abundant disarray of short thin fibrils in comparison with FAK^+/+ controls. (B) FN in wild-type and FAK^-/- E8.5 mouse littermates as seen by immunofluorescence on sections through the headfold. In both embryos the most intense FN staining was between ectodermal and mesodermal layers (arrowheads). The abundance of fibrillar FN matrix was greater in headfold mesoderm of FAK^+/+ mice, whereas in FAK^-/- mice the FN matrix was less fibrillar and more punctate (arrows, black and white images). (C) Immunoelectron microscopy of FN matrix. FN-rich areas on sections of wild-type and FAK^-/- E8.5 mouse embryos. Fn^-/- embryos were used as a negative control. (D) Comparison of FN mRNA levels in embryos from FAK heterozygote crosses. Eight micrograms of total RNA of wild-type (+/+), FAK-heterozygous (+/-), and FAK-null (-/-) genotype was analyzed by northern blot. The membrane was hybridized with radiolabeled mouse FN cDNA probe. 28S and 18S are ribosomal RNAs in an ethidium bromide-stained gel to demonstrate equal loading and quality of RNA. (E) Western blot analysis of FN expression in E8.5 FAK^-/- embryos. E8.5 littermates from FAK^+/- crosses were genotyped, and embryos of the same genotype were pooled and lysed. Slightly stronger FN bands were detected in lysates of FAK^-/- embryos. The absence of FN bands in lysates of Fn^-/- embryos verifies their specificity. Skp1 was used as a loading control.