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Fig. 5. XMeis3 activates FGF/MAPK caudalizing activities. (A) Embryos at the
one-cell stage were injected with 1.0 ng of XMeis3-encoding RNA, 1.0 ng of
FGFI-DNR-encoding RNA or both. Animal cap explants removed at blastula stages
were grown to early and late gastrula stages. Protein was isolated and western
blot analysis of phosphorylated-ERK and total ERK protein was performed. One
representative experiment is shown. (B) The bar graphs for (A) describe
chemiluminescent quantitation of samples performed using an Image Maker VPS-CL
monitor (Amersham-Pharmacia). To quantify the samples, the relative amounts of
phosphorylated ERK and total ERK were calculated and plotted on the bar
graphs. (C) One-cell-stage embryos were injected in the animal hemisphere with
1.0 ng of XMeis3 RNA, 1.0 ng of FGF1 DNR RNA or both. 18
animal cap explants were removed from uninjected and injected groups of
blastula embryos (stage 8-9). Explants from each group were grown to stage 20
and total RNA was isolated. RT-PCR analysis was performed with the markers
Krox20, HoxB9 and HoxD1. EF1
served as a control for
quantifying RNA levels in the different samples. For controls, RT-PCR and
–RT-PCR was performed on total RNA isolated from normal embryos. (D)
One-cell-stage embryos were injected in the animal hemisphere with 1.0 ng
XMeis3 RNA. 36 animal cap explants were removed from uninjected and
injected groups of blastula embryos (stage 8-9). 18 explants from each group
were grown to stages 11.5 and 12.5, and total RNA was isolated. RT-PCR
analysis was performed with the markers Krox20 and FGF3.
EF1 served as a control to quantify RNA levels in the different
samples. For controls, RT-PCR and –RT-PCR (not shown) were performed on
total RNA isolated from normal embryos.
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