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Experiment details for hhex

Li Y et al. (2008) Assay

Sfrp5 coordinates foregut specification and morphogenesis by antagonizing both canonical and noncanonical Wnt11 signaling.

Gene Clone Species Stages Anatomy
hhex.L laevis NF stage 10.5 endoderm , foregut

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  Supplementary Figure 57. Wnt11-MO control experiments. (A) Injection of the wnt11-MO (50 ng) into the dorsal midline at the 4-8-cell stage results in gastrulation defects as previously described (Tada and Smith 2000). (B) In situ hybridization with a cardiac-troponin probe at stage 35. Oorsallateral injection of the wnt11-MO (25ng) into the 4-cell stage often results in reduced cardiac differentiation (Pandur et al. , 2002). When the wnt11-MO was injected into the 01 cells at the 32-cell stage, the cardiac progenitors differentiated but fail to fuse at the midline as previously described (Garriock et al. , 2005). (C) In situ hybridization to bisected gastrula embryos with hhex shows that axial patterning and the organizer were unaffected by injection of the wnt11-MO into 01 cells at the 32-cell stage.

Gene Clone Species Stages Anatomy
hhex.L laevis NF stage 18 liver diverticulum , liver primordium

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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.

Gene Clone Species Stages Anatomy
hhex.L laevis NF stage 18 endoderm , liver diverticulum , liver , foregut , liver primordium

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  Figure 7. Sfrp5 antagonizes endogenous Wnt11 signaling. (A) Reducing endogenous Wnt11 rescues Sfrp5-depleted embryos. Embryos were injected in the D1 anterior endoderm at the 32-cell stage with a wnt11-MO, sfrp5-MO, or both the wnt11-MO and the sfrp5-MO. At stage 18, the foregut epithelium was assayed by anti−β-catenin or aPKC immunostaining of bisected embryos and gene expression was examined by in situ hybridization. Injection of the wnt11-MO expanded hhex and reduced vent2 expression. Coinjection of the wnt11-MO and sfrp5-MO rescued both the epithelial integrity and the changes in gene expression observed with sfrp5-MO alone. (fgc) Foregut cavity. (B) A model of how Sfrp5 restricts both Wnt11/β-catenin and Wnt11/PCP activity to coordinate foregut specification and morphogenesis.

Gene Clone Species Stages Anatomy
hhex.L laevis NF stage 20 endoderm

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  Supplementary Figure 1. Sfrp5 is required for foregut gene expression. (A-C) Gastrula stage hhex expression is unchanged by sfrp5-MO or sfrp5 RNA injection. (D-N) In situ hybridization to bisected stage 20 embryos with the indicated probes. (0 -Q) Cardiac troponin in situ hybridization to stage 35.

Gene Clone Species Stages Anatomy
hhex.L laevis NF stage 20 endoderm , liver diverticulum , foregut

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  Figure 52. Specificity of antisense Sfrp5 depletion (A) Sequence alignment of the Xenopus /aevis sfrp5 mRNA showing the 5' UTR and the start of translation (boxed). Below is the sequence of the sfrp-MO designed to inhibit translation and the negative control sfrp5-mismatch-MO with five nucleotides mutated (red). (B) Examples of control injection and rescue experiments. Embryos were injected in the dorsal-vegetal cells at the 8-cell stage with either the sfrp5-mismatch-MO (50 ng) or the sfrp5-MO (50 ng). For rescue experiments, some of the sfrp5-MO embryos were then injected in the D1 anterior-vegetal cells at the 32-cell stage with sfrp5 RNA (100 pg) lacking the 5' UTR sequence targeted by the MO. Embryos were assayed by in situ hybridization at stages 20 and 35 with hhex and for1 probes respectively. Some embryos were assayed at stage 20 by beta-catenin immunostaining of the foregut epithelium. In all assays the sfrp5-mismatch-MO injected embryos were indistinguishable from the uninjected controls. Co-injection of the sfrp5 RNA robustly rescued the sfrp5-MO phenotype. Higher doses (200-500 pg) caused over expression effects. The number of embryos exhibiting these phenotypes are presented in Fig. 2V and Table S1 .