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mstn is upregulated in LOF Wnt/β-catenin. (A) 2 ng Lef1 MO was injected at 1-cell stage into wild-type or axin1/apc1 embryos, and the number of somites was counted at 54 hpf. Two independent clutches of axin1/apc1 heterozygous incross were analyzed (Total n = 64), in which 10 were genotyped as axin1/apc1 homozygous. (B) Quantitative real-time PCR (qRT-PCR) of myostatin mRNA expression normalized to actin. Total RNA was isolated from 36 hpf wild-type, axin1/apc1 and Lef1 morphant embryos. Graphs show that expression of mstn is upregulated in Lef1 morphants, corresponding to the in situ hybridization with mstn probe in bottom panels. Scale bar, 100 μm. (C) Quantitative real-time PCR (qRT-PCR) of myostatin mRNA expression normalized to actin. Total RNA was isolated from 54 hpf wild-type, axin1/apc1 and Lef1 morphant embryos. Graphs show that expression of mstn is downregulated in axin1/apc1 embryos and upregulated in Lef1 morphants (D) Western blot on lysates collected from 54 hpf wild-type, axin1/apc1 and Lef1 morphant embryos. Graph shows upregulation of Mstn in Lef1 morphants. However, there is no significant difference in levels of Mstn protein in axin1/apc1 embryos versus wildtypes. (E) Misexpression of mstn mRNA downregulates the Wnt target gene lef1 in axin1/apc1 mutants shown with WISH for lef1 riboprobe. Scale bar, 100 μm.
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