Fig. 2. Ablation of integrin β1 in embryoid bodies (EBs) disrupts endoderm morphogenesis. Wild-type (WT) and integrin-β1-null (β1–/–) EBs were cultured in suspension for 2-7 days. (A) Live phase micrographs show endoderm (en) formation on day 3, and epiblast (epi) polarization and cavitation (cv) on days 5 and 7, respectively, in wild-type EBs (arrows). By contrast, endoderm cells detached from or loosely adhered to the EB surface (arrowheads) in most of the mutant EBs. BM, basement membrane. (B) 5-day-old EBs were immunostained for the endoderm markers -fetoprotein (-FP), cytokeratin EndoA, GATA4 and disabled-2 (Dab2). BM is identified by laminin 1/1 chain (Lm 1/1) or perlecan (perl) immunofluorescence. Arrowheads point to positively stained endoderm cells. (C) EBs that had developed a continuous -FP-positive endoderm layer were counted and plotted as a percentage of total EBs examined. More than 85% of the WT EBs developed a continuous endoderm layer, whereas less than 4% of the mutant EBs had nearly normal endoderm. (D) Western analysis was performed for the expression of endoderm markers on 5-day-old normal and mutant EBs collected together with detached endoderm cells. Actin serves as loading control. (E) Normal endoderm cells were harvested by brief trypsinization and mechanical dissociation. The detached integrin-β1-null endoderm cells were collected from the conditioned medium. The expression of the BM proteins laminin-111, nidogen, collagen IV (Col IV) and fibronectin (Fn) was analyzed by western blotting.