Fig. 2. Expression of Dlk in immature liver cells in vivo and in vitro. RT-PCR (A) and northern blotting (B,C) were performed to detect Dlk mRNA during liver development and fetal hepatocyte primary culture. (A) Dlk expression was clearly detected in the E10.5 liver bud and the E14.5 liver but not in the neonatal liver shown by RT-PCR using cDNA synthesized from total RNA of livers at each stage. GAPDH expression was also examined to ensure an equal quantity of cDNA used for PCR. (B) Dlk was strongly expressed in fetal livers between E12.5 and E16.5, but it was rapidly downregulated in later gestation. In neonatal and adult livers, its expression was not detected. Each lane was loaded with 10 µg of total RNA extracted from livers at each stage. GAPDH expression was also examined to ensure an equal loading of RNA. (C) Fetal hepatic cells were prepared from E14.5 fetal liver and cultured on gelatin-coated dishes in the presence or absence of dexamethasone (Dex) and oncostatin M (OSM). TAT and CPS were weakly expressed after 4 days and clearly detected after 7 days of culture with Dex and OSM. Each lane was loaded with 10 µg of total RNA extracted from cultured cells at each time point. Note that Dlk was rapidly downregulated in the presence of Dex and OSM, although downregulation occurred spontaneously without OSM.