Fig. 3. In vitro explant culture assay for PE marker induction. (A) Schematic of the assay. Chick lateral embryonic fragments (cLEF) were isolated from the fourth to seventh (4-7) somite levels of stage 10-11 embryos. Width of the somite(s) was used as a morphological reference for mediolateral levels of the fragment. The fragment was cultured alone, or co-cultured with the quail liver bud (qLiB) or lung bud (qLuB) in a hanging drop of M199. (B) Reverse transcriptase (RT)-PCR analysis of mRNA isolated from cultured explants. Weak signals for Wt1, capsulin and Pax2 are detectable in cLEF cultured alone. The level of signals for Wt1 and capsulin, but not Pax2, increased significantly when cLEF was co-cultured with the liver bud. In our RT-PCR condition, mRNAs of all these markers were undetectable in the liver bud cultured alone. (C) Quantification of PCR products of Wt1, capsulin and Pax2, showing enhancement of proepicardium (PE) marker expression in co-culture with the liver bud. Standard deviation bars are shown. (D) RT-PCR analysis using chick-specific primers for Wt1 (cWt1), Tbx18 (cTbx18) and Cfc1 (cCfc1). The liver bud has a strong capacity to upregulate PE marker gene expression in co-cultured cLEF. (E) Quantification of PCR products. We performed PCR changing the amount of the template cDNA to ensure linear amplification conditions. (F) Quantitative real-time (qRT)-PCR analysis of cWt1. Bars show the average of three independent PCR reactions.