Fig. 1. Arnt-null TS cells differentiate into chorionic trophoblasts and syncytiotrophoblasts. Undifferentiated wild-type (A) and Arnt-null (B) TS cells grown on fibroblast feeders exhibited identical morphological features. Differentiated wild-type cells (C) formed large trophoblast giant cells and smaller spongiotrophoblasts. Differentiated Arnt-null TS cells (D) produced large multinucleated syncytiotrophoblasts and smaller chorionic trophoblasts. E-cadherin staining of differentiated wild-type cells revealed one or occasionally two nuclei per cell (E), whereas Arnt-null cells formed syncytia containing multiple nuclei (F). In some cases, multinucleated cells with peripherally distributed nuclei (G, arrowheads) and central clearing appeared to form epithelial-lined spaces. (H) RNA FISH analysis of Xist RNA revealed a single inactive X chromosome per nucleus in differentiated Arnt-null TS cells, additional evidence for lack of endoreduplication. (I) Analysis of lineage-specific markers by northern blot hybridization and RNase protection assays confirmed these findings.