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Fig. 4
fscn1 regulates endoderm formation and Nodal signal transduction via its actin-bundling activity.
(a-c) The expression of endoderm markers sox32 and gata5 in wild-type embryos and embryos treated with Lat A. Embryos were treated with 0.1 µM Lat A from the sphere stage to shield stage (a), and analysed by in situ hybridization (b) and real-time PCR (c) for sox32 and gata5 expression. In c, the expression of ²-actin was used as a reference to normalize the amount of mRNAs in each sample. The data are presented as mean ±s.d. of three independent experiments. Student’s t test, ***P<0.001. (d) Actin filaments regulate Nodal/Smad2 signal transduction. Wild-type embryos were treated with 0.1 µM Lat A from the sphere to shield stages and then harvested for immunoblotting. Quantification is the relative density of phospho-specific signals to corresponding total protein signals (mean±s.d., three independent biological repeats). (e-g) Overexpression of Fscn1 S39A, but not S39D, recovers the expression reduction of ARE-luciferase reporters and endodermal markers in fscn1a morphants and MZfscn1a mutants. Embryos were co-injected with the indicated MOs, plasmids and mRNAs at the one-cell stages and collected at the 75% epiboly stage for luciferase measurement (e) and in situ hybridization (f,g). The relative luciferase activity was the mean with s.d. from three independent experiments. Injection doses: ARE-luc, 100 pg; MO1, 4 ng; wild-type fscn1 mRNA, 150 pg; S39A mRNA, 150 pg; S39D mRNA, 150 pg. Student’s t test, ***P<0.001. (h) The expression of sox32 and sox17 were examined at the 75% epiboly stage in embryos injected with different dose of pkcα mRNA. (i) Fscn1 S39A overexpression antagonizes the PKCα-induced decrease of endoderm formation. One-cell stage embryos were co-injected with 400 pg pkcα mRNA and 150 pg Fscn1 S39A or S39D mRNA, then collected at the 75% epiboly stage for in situ hybridization and quantification of endodermal marker sox17 expression. Scale bar, 200 µm.