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Figure 6. Sfrp5 coordinates foregut morphogenesis and specification by restricting noncanonical Wnt/JNK and canonical Wnt/β-catenin, respectively. (A) Membrane-localized Dsh is elevated in Sfrp5-depleted foregut cells. RNA encoding myc-tagged Dsh was injected into the anterior endoderm of control or sfrp5-MO-injected embryos, and its subcellular localization was determined by anti-myc immunostaining at stage 18. In control embryos, membrane-localized Dsh-myc was observed in the deep endoderm cells (yellow arrow) but not in the surface epithelium next to the foregut cavity (fgc). Foci of membrane-localized Dsh-myc were detected in the dissociating surface cells of Sfrp5-depleted foreguts (white arrows). (B) Depletion of sfrp5 results in a specific increase in β-catenin/Tcf and JNK/AP1 activity in the foregut. TOP: flash or AP1:Luciferase reporter plasmids were injected into either the D1 foregut endoderm cells or the D4 hindgut endoderm cells at the 32-cell stage of control or Sfrp5-depleted embryos. The TOP:Flash reporter is an indicator of β-catenin/Tcf activity, while the AP1:luciferase reporter is an indicator of JNK activation of c-Jun, a component of the AP1 complex. At stage 20, the reporter activity was determined by luciferase assays, in triplicate. The average values normalized to coinjected pRTK:Renila + standard deviation. (*) P < 0.05 in Student t-test compared with control foreguts. (C) JNK activity is elevated in Sfrp5-depleted foreguts. Foregut explants were isolated from controls, sfrp5-MO-injected embryos, or embryos treated with a JNK inhibitor (SP600125). The Western blot shows the results of a phospho-c-jun JNK activity assay. (D) Sfrp5 inhibits Wnt/β-catenin signaling to maintain foregut gene expression, and inhibits Wnt/PCP signaling to maintain foregut epithelial integrity. The indicated constructs were injected into the D1 foregut endoderm cells at the 32-cell stage. At stage 18, bisected embryos were assayed by anti-β-catenin immunostaining or by hhex and for1 in situ at stages 18 and 35, respectively. (E) Representative examples are shown, and a summary is presented in the graph below. A complete summary of all the rescue experiments and controls is presented in Supplemental Table S1. |
