Fig. 5. Cytotrophoblast migration is reduced by co-culture with 3T3 cells that express the venous receptor EPHB4. (A) Staining of stably transfected 3T3 cell lines with antibodies specific for EPHB4 and ephrin B2 showed that they expressed the respective proteins. (B,D) Our previous work (Librach et al., 1991) showed that isolated cytotrophoblasts cultured on laminin or Matrigel substrates rapidly migrate toward one another, forming numerous aggregates. Similar behavior was observed when the cytokeratin-positive (CK7) cytotrophoblasts (green) were co-cultured with control 3T3 cells. (C,E) By contrast, when cytotrophoblasts were cultured with 3T3 cells expressing EPHB4 (3T3-EPHB4), aggregate formation was markedly inhibited, and the cells appeared to spread on the substrate. Tracking cell movement for 15 hours showed that, in comparison with controls (F), cytotrophoblasts co-cultured with 3T3-EPHB4 cells migrated much shorter distances (G). (H) Quantifying the average linear distance traveled showed that migration was significantly reduced when the cytotrophoblasts were co-cultured with 3T3-EPHB4 cells on either laminin or Matrigel substrates. Error bars indicate s.d. Scale bars: 20 µm in A; in B, 50 µm for B,C; in D, 10 µm for D,E.