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Satoshi Komiya^1,2,, Masayuki Shimizu^1,, Junichi Ikenouchi^3,, Shigenobu Yonemura⁴, Takeshi Matsui⁵, Yoshitaka Fukunaga¹, Huijie Liu¹, Fumio Endo², Shoichiro Tsukita³ and Akira Nagafuchi^1,*

¹ Division of Cellular Interactions, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan
² Department of Pediatrics, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto 860-8556, Japan
³ Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan
⁴ Laboratory for Cellular Morphogenesis, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan
⁵ KAN Research Institute, Inc., Shimogyo-ku, Kyoto 600-8815, Japan

Epithelium formation is a common event in animal morphogenesis. It has been reported that F9 cells differentiate into visceral endoderm-like epithelial cells when cell aggregates are cultured in the presence of retinoic acid. The present investigation set out to determine whether this in vitro model could be used under monolayer culture conditions, which is suitable for a detailed analysis of epithelial differentiation. We performed comparative gene expression analyses of F9 cells grown under aggregate and monolayer culture conditions prior to and following treatment with retinoic acid. Under these conditions, induction in the expression of differentiation marker genes was confirmed, even in monolayer cultures. Junctional complex and apical membrane formation, both of which are characteristic of epithelial cells, were also observed under monolayer culture conditions. Because of the merit of monolayer culture condition, we found that apical membrane and junctional complex formation are strictly regulated during epithelial differentiation. It was also revealed that F9 cells differentiated into epithelial cells predominantly on the fourth and fifth day following retinoic acid induction. These results showed that a monolayer culture of F9 cells represents a viable in vitro model that can be employed to elucidate mechanisms pertaining to epithelium formation.

These authors contributed equally to this work.

^* Correspondence: E-mail: naga-san{at}kaiju.medic.kumamoto-u.ac.jp

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