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Fig. 3. Expression analysis of Cer-EGFP reporter constructs.
(A-H) Cer-EGFP reporter expression in electroporated chick
embryos. Different embryos were co-transfected with one Cer-EGFP
reporter construct (green fluorescence) and the pCAGGS-RFP construct
(positive control; red fluorescence). (A,C) Cer0.4-EGFP; (B,D) F2mut; (E)
PCR3; (F) PCR2; (G) FSF; (H) FF (see Fig.
2 for construct details). (A,B) EGFP fluorescence was observed in
the anterior mesendoderm (AM) of embryos electroporated with Cer0.4 (A) and
F2mut (B) reporter constructs. Embryos were electroporated at Hamburger and
Hamilton stage 3 (HH3) and fixed at HH6. (C-H) Asymmetric EGFP
expression was detected in the left-side mesoderm (LSM) of embryos
electroporated with Cer0.4 (C), PCR3 (E) and FSF (G), but not in those
electroporated with the F2mut (D), PCR2 (F) or FF (H) reporter constructs.
Embryos were electroporated at HH4-5 and fixed at HH8-9. Dashed line separates
the right and left sides of the embryos. (I,J) Cer-Luc
reporter activity in Xenopus animal cap luciferase assays. Luciferase
reporter plasmids containing the indicated wild-type or mutant fragments of
chick Cer (cCer) regulatory sequences were injected into
Xenopus embryos in the absence (orange) or presence (green) of
Nodal mRNA. Data are relative to the highest luciferase activity
values (Cer0.36+Nodal in I; PCR3+Nodal and FSF+Nodal in J). The activities of
reporter constructs that either lack one of the FoxH1 elements (F1mut, F2mut,
PCR1, PCR2 or FS) or lack the SMAD element (Smut and FF) were reduced. AM,
anterior mesendoderm; L, left; LSM, left-side mesoderm; R, right.
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