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Fig. 4. FoxH1 depletion does not prevent animal caps from responding to activin or
Xnr1. (A) Groups of ten control or FoxH1-depleted animal caps were dissected
at the mid-blastula stage, treated with activin (1 µg/ml) for 4 hours and
frozen at the early gastrula stage and assayed for expression of nodal target
genes including Mix.2, Fgf8, Xbra, goosecoid, chordin, Xbra and
Xnr1. In the same experiment, Xnr1 mRNA (2 pg), was injected
into wild-type or FoxH1-depleted embryos at the two-cell stage, and caps
dissected as above. Expression levels were compared with one wild-type embryo
at the early gastrula stage (control we). No significant changes in activin
and nodal target gene expression were seen in FoxH1-depleted caps compared to
control caps. (B,C,D) In one experiment, the degree of reduction of
ARE-luciferase activity (B), and the level of induction of nodal-response
genes in sibling animal caps (C) was measured at the gastrula stage, and the
phenotype of sibling embryos was examined (D) at the tailbud stage. (B)
FoxH1-depleted caps are unable to activate ARE-luciferase in response to
activin (1 µg/ml). (C) Sibling FoxH1-depleted caps respond normally to
Xnr1 mRNA (2 pg) injected at the two-cell stage by expressing
Mix.2, chordin, Fgf8, Xbra, Xnr2 and goosecoid as measured
by real time RT-PCR. (D) Sibling embryos (right) develop with a headless
phenotype compared to controls (three embryos on the left). (E) The expression
of mesodermal and endodermal early zygotic genes analysed by real-time RT-PCR
in a temporal series of control and FoxH1-depleted embryos, frozen at the
blastula and gastrula stages. All of these results were repeated in a second
experiment. Most endodermal and mesodermal genes show some reduction of
expression. Xnr5 and 6 showed increased expression compared
to control levels.
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