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nodal5xenopus ectoderm [+] 

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Experiment details for nodal5

Klf4 is required for germ-layer differentiation and body axis patterning during Xenopus embryogenesis.

Klf4 is required for germ-layer differentiation and body axis patterning during Xenopus embryogenesis.

Gene Clone Species Stages Anatomy
nodal5.L laevis NF stage 8 to NF stage 9 ectoderm

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  Fig. 4. The regulatory effect of Klf4 on the Nodal/Activin pathway. (A-C) The effect of Klf4 overexpression on transcription of Xnr1 (A), Xnr5 (B) and Mix2 (C). Klf4 mRNA (300 pg) was injected into the equatorial region of all blastomeres at the four-cell stage. Control and injected embryos at stage 8.5 were collected for detection of Xnr1 and Xnr5 expression, while embryos at stage 10 were collected for detection of Mix2 expression. In A and B, embryos were in lateral view. In C, vegetal view was shown for ‘ctrl’ to reveal normal expression of Mix2, and animal view was shown for ‘Klf4’ to reveal ectopic Mix2 expression in ectoderm. (D) Quantification of embryos in A-C in three experiments. (E) Luciferase assays in HEK293T cells transfected with ARELuc reporter plasmid and expression plasmids for Xnr5, FAST1 or Klf4. (F) Luciferase assays in embryos injected with ARELuc reporter plasmid and mRNAs for Xnr2 or Klf4. (G) Luciferase assays on pGL3-basic vector in embryos injected with Xnr2 or Klf4 mRNAs. Error bars represent s.d. in eight experiments. In F and G, plasmids were injected at 40 pg, Xnr2 mRNA was injected at 10 pg, and Klf4 mRNA was injected at 300 pg. (H) Mix2 expression in uninjected (Uninj.) and Klf4 RNA-injected (Klf4) embryos, and treated separately with different chemicals as indicated. All embryos are in vegetal view. Klf4 mRNA (400 pg) was injected vegetally at the four-cell stage, treated with chemicals and collected at stage 10.5 for whole-mount in situ hybridization. (I) Quantification of embryos in H with normal, downregulated or upregulated Mix2 expression in three experiments. (J) Mix2Luc(−712) luciferase assay with untreated embryos or embryos treated with SB431542. Plasmid was injected at 40 pg, Xnr2 mRNA was at 10 pg, and Klf4 mRNA was injected at 300 pg. Error bars represent s.d. in seven experiments. (K) The rescuing effect of Klf4 on Mix2 expression. All embryos were in vegetal view. dnXAR1 RNA (1.5 ng) and Klf4 RNA (400 pg) were injected separately or together into the vegetal pole of four-cell embryos. Embryos were collected at stage 11 for whole-mount in situ hybridization. (L) Quantification of embryos with normal or altered Mix2 expression observed in K. Error bars represent s.d. in triplicate. Student's t-test showed the significance of the ratio of rescued Mix2 expression in embryos with dnXAR1+Klf4 RNA injection compared with background (ratio of embryos with unaffected Mix2 expression in dnXAR1-injected embryos). Asterisks indicate P<0.01.