|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
|
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.
|
