During early rodent development, the parietal endoderm appears from an inner cell mass and produces large amounts of basement membrane components, such as laminin-1 and collagen IV. To elucidate the regulatory network for gene expression during these procedures, we constructed a series of short interfering RNA expression vectors targeted to various transcription factors, transfected them into F9 embryonal carcinoma cells, and evaluated the effects of the gene silencing on the induction of parietal endoderm differentiation and basement membrane component production by treating F9 cells with all trans-retinoic acid and dibutyryl cyclic AMP. Among the transcription factors tested, silencing of Sox7 or combined silencing of Gata-4 and Gata-6 resulted in suppression of cell shape changes and laminin-1 production, which are the hallmarks of parietal endoderm differentiation. In cells silenced for Sox7, induction of Gata-4 and Gata-6 by retinoic acid and cyclic AMP treatment was inhibited, while induction of Sox7 was not affected in cells silenced for Gata-4 and Gata-6, indicating that Sox7 is an upstream regulatory factor for these Gata factors. Nevertheless, silencing of Sox7 did not totally cancel the action of retinoic acid, since upregulation of coup-tf2, keratin 19, and retinoic acid receptor ␤2 was not abolished in Sox7-silenced F9 cells. Although overexpression of Sox7 alone was insufficient to induce parietal endoderm differentiation, overexpression of Gata-4 or Gata-6 in Sox7-silenced F9 cells restored the differentiation into parietal endoderm. Sox7 is therefore required for the induction of Gata-4 and Gata-6, and the interplay among these transcription factors plays a crucial role in parietal endoderm differentiation.

Basement membranes (BMs) have been implicated in cell fate determination during development. Embryoid bodies (EBs) derived from mouse embryonic stem cells deficient in the laminin ␥1 chain are incapable of depositing a BM, resulting in failure of primitive ectoderm epithelialization. To elucidate the mechanisms involved in this phenomenon, we compared the gene expression profiles of EBs with or without a BM to identify the genes showing BM-dependent expression. We found that the expressions of marker genes for the epithelial-mesenchymal transition (EMT), including the transcription factor Snai2, were up-regulated in LAMC1 ؊/؊ EBs, whereas restoration of a BM to LAMC1 ؊/؊ EBs suppressed the up-regulation of these genes. Overexpression of Snai2 induced the EMT in control EBs by molecular and morphological criteria, suggesting that suppression of the EMT regulatory genes is involved in BM-dependent epithelialization of primitive ectoderm. Despite the failure of primitive ectoderm epithelialization in BM-deficient EBs, mesodermal differentiation was not compromised, but rather accelerated. Furthermore, at later stages of control EB differentiation, the BM was disrupted at the gastrulation site where mesodermal markers were strongly expressed only in cells that had lost contact with the BM. Taken together, these results indicate that the BM prevents the EMT and precocious differentiation of primitive ectoderm toward mesoderm in EBs, implying that BMs are important for the control of mammalian gastrulation.

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