Apical membrane and junctional complex formation during simple epithelial cell differentiation of F9 cells

Abstract

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.

Retinoic Acid Induces Expression of the Thyroid Hormone Transporter, Monocarboxylate Transporter 8 (Mct8)

“…tRA treatment induced primitive/visceral endoderm differentiation markers, Col4a1, Lama1, and Afp, in F9 cells, whereas the combination with tRA and 8-bromo-cAMP induced parietal endoderm differentiation markers, Col4a1, Lama1, and Thbd (Fig. 3, A-C), consistent with previous reports (13,14). The expression of Mct8 mRNA was markedly increased with any extraembryonic endoderm differentiation induced, although the addition of 8-bromocAMP modestly reduced the magnitude of induction (Fig.…”

“…These data indicate that, among the thyroid hormone transporter genes tested in F9 cells, only primitive, parietal, and visceral (13,14). Combination treatment with tRA and cAMP, under low serum conditions, induces morphologically neuron-like cells with neuron-specific acetylcholinesterase activity (15).…”

“…Briefly, total RNA from culture cells was prepared with the RNeasy minikit (Qiagen, Valencia, CA) with on-column DNase digestion. Three g of total RNA was reverse-transcribed by using 50 units of Superscript III reverse transcriptase (Invitrogen) in a 20-l reaction with oligo-(dT) [12][13][14][15][16][17][18] primer or random hexamer. Quantitative PCR of mouse thyroid hormone transporter genes, glyceraldehyde-3-phosphate dehydrogenase gene (Gapdh), 18 S ribosomal RNA, and human MCT8 mRNA was performed with custom DNA primers synthesized by Invitrogen (supplemental Table 1).…”

“…A combination treatment of F9 cells with all-trans-RA (tRA), an agonist of RAR, and cAMP induces extraembryonic endoderm (13,14) or neuron-like cells (15), depending on the composition of culture media. The extraembryonic endoderm supports the developing embryo and facilitates exchange of small molecules between the maternal circulation and the embryo, functioning as an "early placenta" (16).…”

Deficiency of Zonula Occludens-1 Causes Embryonic Lethal Phenotype Associated with Defected Yolk Sac Angiogenesis and Apoptosis of Embryonic Cells

“…Mouse and rat anti-ZO-1 monoclonal antibody (mAb) (Ioth et al, 1993;Kitajiri et al, 2004), rabbit anti-ZO-3 polyclonal antibody (pAb) (Inoko et al, 2003), rat anti-occludin mAb (Saitou et al, 1997), and rabbit anti-junctional adhesion molecule (JAM)-A pAb (Komiya et al, 2005), rabbit anti-claudin-6 pAb and rat anti-cingulin mAb (Ohnishi et al, 2004) were used as described previously. Rat anti-mouse E-cadherin mAb (ECCD2) was generously provided by Dr. M. Takeichi (Center for Developmental Biology, Kobe, Japan).…”