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Fig. 3. Endodermal detachment and disorganized F-actin in PINCH1-null EBs. (A,B) Phase-contrast images of wild-type EB (A) showing the epiblast layer consisting of pseudostratified columnar epithelial cells, and PINCH1-null EB (B) showing a multi-layered and polygonal epiblast cell layer sometimes associated with incomplete cavitation. (C,D) Immunofluorescence of PINCH1 (green) and laminin {gamma}1 (red) revealed PINCH1 expression at the basement membrane zone of normal endoderm and epiblast and in the nucleus of some endodermal cells (C). PINCH1-null EBs lack PINCH1 signals but show a linear laminin {gamma}1 staining (D). Nuclei are counterstained with DAPI (blue). (E,F) ß1 integrin (green) is condensed in the basal plasma membranes of normal endoderm and epiblast where it colocalizes with laminin {gamma}1 (red; arrowheads; E). Signals for ß1 integrin are similarly strong in PINCH1-null EBs (F). (G,H) F-actin (red) forms an apical belt and bundles parallel to lateral plasma membranes in the normal, polarized epiblast (G). PINCH1-null epiblast contains actin aggregates at the basal plasma membranes and lacks the parallel actin bundles (H).





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